Interleukin-17 receptor homologue

ABSTRACT

Cytokines and their receptors have proven usefulness in both basic research and as therapeutics. The present invention provides a new human cytokine receptor designated as &#34;Zcytor14.&#34;

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/458,647, filed Jun. 10, 2003, which is a continuation of U.S.application Ser. No. 09/608,918, filed Jun. 30, 2000, which claims thebenefit of U.S. Provisional Application Ser. No. 60/142,555, filed Jul.7, 1999, both of which are herein incorporated by reference.

TECHNICAL FIELD

The present invention relates generally to a new protein expressed byhuman cells. In particular, the present invention relates to a novelgene that encodes a receptor, designated as “Zcytor14,” and to nucleicacid molecules encoding Zcytor14 polypeptides.

BACKGROUND OF THE INVENTION

Cytokines are soluble, small proteins that mediate a variety ofbiological effects, including the regulation of the growth anddifferentiation of many cell types (see, for example, Arai et al., Annu.Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol. 3:311 (1991);Paul and Seder, Cell 76:241 (1994)). Proteins that constitute thecytokine group include interleukins, interferons, colony stimulatingfactors, tumor necrosis factors, and other regulatory molecules. Forexample, human interleukin-17 is a cytokine that stimulates theexpression of interleukin-6, intracellular adhesion molecule 1,interleukin-8, granulocyte macrophage colony-stimulating factor, andprostaglandin E2 expression, and plays a role in the preferentialmaturation of CD34+ hematopoietic precursors into neutrophils (Yao etal., J. Immunol. 155:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593(1996)).

Receptors that bind cytokines are typically composed of one or moreintegral membrane proteins that bind the cytokine with high affinity andtransduce this binding event to the cell through the cytoplasmicportions of the certain receptor subunits. Cytokine receptors have beengrouped into several classes on the basis of similarities in theirextracellular ligand binding domains. For example, the receptor chainsresponsible for binding and/or transducing the effect of interferons aremembers of the type II cytokine receptor family, based upon acharacteristic 200 residue extracellular domain.

The demonstrated in vivo activities of cytokines and their receptorsillustrate the clinical potential of, and need for, other cytokines,cytokine receptors, cytokine agonists, and cytokine antagonists.

SUMMARY OF THE INVENTION

The present invention provides a novel receptor, designated “Zcytor14.”The present invention also provides Zcytor14 polypeptides and Zcytor14fusion proteins, as well as nucleic acid molecules encoding suchpolypeptides and proteins, and methods for using these nucleic acidmolecules and amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

An illustrative nucleotide sequence that encodes Zcytor14 is provided bySEQ ID NO:1. The encoded polypeptide has the following amino acidsequence: MPVPWFLLSL ALGRSPVVLS LERLVGPQDA THCSPGLSCR LWDSDILCLPGDIVPAPGPV LAPTHLQTEL VLRCQKETDC DLCLRVAVHL AVHGHWEEPE DEEKFGGAADSGVEEPRNAS LQAQVVLSFQ AYPTARCVLL EVQVPAALVQ FGQSVGSVVY DCFEAALGSEVRIWSYTQPR YEKELNHTQQ LPALPWLNVS ADGDNVHLVL NVSEEQHFGL SLYWNQVQGPPKPRWHKNLT GPQIITLNHT DLVPCLCIQV WPLEPDSVRT NICPFREDPR AHQNLWQAARLRLLTLQSWL LDAPCSLPAE AALCWRAPGG DPCQPLVPPL SWENVTVDKV LEFPLLKGHPNLCVQVNSSE KLQLQECLWA DSLGPLKDDV LLLETRGPQD NRSLCALEPS GCTSLPSKASTRAARLGEYL LQDLQSGQCL QLWDDDLGAL WACPMDKYIH KRWALVWLAC LLFAAALSLILLLKKDHAKA AARGRAALLL YSADDSGFER LVGALASALC QLPLRVAVDL WSRRELSAQGPVAWFHAQRR QTLQEGGVVV LLFSPGAVAL CSEWLQDGVS GPGAHGPHDA FRASLSCVLPDFLQGRAPGS YVGACFDRLL HPDAVPALFR TVPVFTLPSQ LPDFLGALQQ PRAPRSGRLQERAEQVSRAL QPALDSYFHP PGTPAPGRGV GPGAGPGAGD GT (SEQ ID NO:2).

Thus, the Zcytor14 gene encodes a polypeptide of 692 amino acids.Features of Zcytor14 include a putative signal sequence (amino acidresidues 1 to 20 of SEQ ID NO:2), an extracellular domain (amino acidresidues 21 to 452 of SEQ ID NO:2), a transmembrane domain (amino acidresidues 453 to 473 of SEQ ID NO:2), and an intracellular domain(comprising amino acid residues 474 to 677 of SEQ ID NO:2).

A variant Zcytor14 protein, designated as “Zcytor14-1,” was identified,which has the following amino acid sequence: EEPRNASLQA QVVLSFQAYPTARCVLLEVQ VPAALVQFGQ SVGSVVYDCF EAALGSEVRI WSYTQPRYEK ELNHTQQLPALPWLNVSADG DNVHLVLNVS EEQHFGLSLY WNQVQGPPKP RWHKNLTGPQ IITLNHTDLVPCLCIQVWPL EPDSVRTNIC PFREDPRAHQ NLWQAARLRL LTLQSWLLDA PCSLPAEAALCWRAPGGDPC QPLVPPLSWE NVTVDVNSSE KLQLQECLWA DSLGPLKDDV LLLETRGPQDNRSLCALEPS GCTSLPSKAS TRAARLGEYL LQDLQSGQCL QLWDDDLGAL WACPMDKYIHKRWALVWLAC LLFAAALSLI LLLKKDHAKG WLRLLKQDVR SGAAARGRAA LLLYSADDSGFERLVGALAS ALCQLPLRVA VDLWSRRELS AQGPVAWFHA QRRQTLQEGG VVVLLFSPGAVALCSEWLQD GVSGPGAHGP HDAFRASLSC VLPDFLQGRA PGSYVGACFD RLLHPDAVPALFRTVPVFTL PSQLPDFLGA LQQPRAPRSG RLQERAEQVS RALQPALDSY FHPPGTPAPGRGVGPGAGPG AGDGT (SEQ ID NO:5). An illustrative nucleotide sequence thatencodes this polypeptide is provided by SEQ ID NO:4.

Sequence analysis revealed that Zcytor14-1 is a truncated form ofreceptor polypeptide. That is, Zcytor14-1 lacks amino acid residues1-113 of SEQ ID NO:2. SEQ ID NO:10 presents an amino acid sequence of aZcytor14-1 polypeptide that includes the N-terminal portion of Zcytor14.

A comparison of the Zcytor14 and Zcytor14-1 amino acid sequences alsoindicated that the two polypeptides represent alternatively splicedvariants. The amino acid sequence of Zcytor14 includes a 17 amino acidsegment (amino acid residues 339 to 355 of SEQ ID NO:2), whichZcytor14-1 lacks, while Zcytor14 lacks, following amino acid 479, a 13amino acid segment found in Zcytor14-1 (amino acid residues 350 to 362of SEQ ID NO:5). A polypeptide that contains both amino acid segments isprovided by SEQ ID NO:11, whereas SEQ ID NO:12 presents the amino acidsequence of a polypeptide that lacks both 13 and 17 amino acid segments.

The Zcytor14 gene resides in chromosome 3p25-3p24. As discussed below,this region is associated with various disorders and diseases.

Northern analyses indicate that there is strong expression of theZcytor14 gene in thyroid, adrenal gland, prostate, and liver tissues,and less expression in heart, small intestine, stomach, and tracheatissues. In contrast, there is little or no expression in brain,placenta, lung, skeletal muscle, kidney, pancreas, spleen, thymus,testis, ovary, colon, peripheral blood leukocytes, spinal cord, lymphnode, and bone marrow. These observations show that Zcytor14 sequencescan be used differentiate between various tissues.

As described below, the present invention provides isolated polypeptidescomprising an amino acid sequence that is at least 70%, at least 80%, orat least 90% identical to a reference amino acid sequence selected fromthe group consisting of: (a) amino acid residues 21 to 452 of SEQ IDNO:2, (b) amino acid residues 21 to 435 of SEQ ID NO:10, (c) amino acidresidues 21 to 677 of SEQ ID NO:2, and (d) amino acid residues 1 to 692of SEQ ID NO:2, wherein the isolated polypeptide specifically binds withan antibody that specifically binds with a polypeptide consisting ofeither the amino acid sequence of SEQ ID NO:2, or the amino acidsequence of SEQ ID NO:10. Illustrative polypeptides include apolypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ IDNO:10, SEQ ID NO:11, or SEQ ID NO:12.

The present invention also provides isolated polypeptides comprising anextracellular domain, wherein the extracellular domain comprises eitheramino acid residues 21 to 452 of the amino acid sequence of SEQ ID NO:2or amino acid residues 21 to 435 of the amino acid sequence of SEQ IDNO:10. Such polypeptides may further comprise a transmembrane domainthat resides in a carboxyl-terminal position relative to theextracellular domain, wherein the transmembrane domain comprises aminoacid residues 453 to 473 of SEQ ID NO:2. These polypeptides may alsocomprise an intracellular domain that resides in a carboxyl-terminalposition relative to the transmembrane domain, wherein the intracellulardomain comprises either amino acid residues 474 to 677 of SEQ ID NO:2,or amino acid residues 457 to 673 of SEQ ID NO:10, and optionally, asignal secretory sequence that resides in an amino-terminal positionrelative to the extracellular domain, wherein the signal secretorysequence comprises amino acid residues 1 to 20 of the amino acidsequence of SEQ ID NO:2.

The present invention also includes variant Zcytor14 polypeptides,wherein the amino acid sequence of the variant polypeptide shares anidentity with the amino acid sequence of SEQ ID NO:2 selected from thegroup consisting of at least 70% identity, at least 80% identity, atleast 90% identity, at least 95% identity, or greater than 95% identity,and wherein any difference between the amino acid sequence of thevariant polypeptide and the amino acid sequence of SEQ ID NO:2 is due toone or more conservative amino acid substitutions.

The present invention further provides antibodies and antibody fragmentsthat specifically bind with such polypeptides. Exemplary antibodiesinclude polyclonal antibodies, murine monoclonal antibodies, humanizedantibodies derived from murine monoclonal antibodies, and humanmonoclonal antibodies. Illustrative antibody fragments include F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv, and minimal recognition units. The presentinvention further provides compositions comprising a carrier and apeptide, polypeptide, antibody, or anti-idiotype antibody describedherein.

The present invention also provides isolated nucleic acid molecules thatencode a Zcytor14 polypeptide, wherein the nucleic acid molecule isselected from the group consisting of: (a) a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:3, (b) a nucleic acidmolecule encoding an amino acid sequence that comprises either aminoacid residues 21 to 677 of SEQ ID NO:2 or amino acid residues 21 to 673of SEQ ID NO:10, and (c) a nucleic acid molecule that remains hybridizedfollowing stringent wash conditions to a nucleic acid moleculecomprising the nucleotide sequence of nucleotides 214 to 2184 of SEQ IDNO:1, or the complement of nucleotides 214 to 2184 of SEQ ID NO:1.Illustrative nucleic acid molecules include those in which anydifference between the amino acid sequence encoded by nucleic acidmolecule (c) and the corresponding amino acid sequence of SEQ ID NO:2 isdue to a conservative amino acid substitution. The present inventionfurther contemplates isolated nucleic acid molecules that comprisenucleotides 214 to 2184 of SEQ ID NO:1 or nucleotides 154 to 2184 of SEQID NO:1.

The present invention also includes vectors and expression vectorscomprising such nucleic acid molecules. Such expression vectors maycomprise a transcription promoter, and a transcription terminator,wherein the promoter is operably linked with the nucleic acid molecule,and wherein the nucleic acid molecule is operably linked with thetranscription terminator. The present invention further includesrecombinant host cells and recombinant viruses comprising these vectorsand expression vectors. Illustrative host cells include bacterial,yeast, fungal, insect, mammalian, and plant cells. Recombinant hostcells comprising such expression vectors can be used to produce Zcytor14polypeptides by culturing such recombinant host cells that comprise theexpression vector and that produce the Zcytor14 protein, and,optionally, isolating the Zcytor14 protein from the cultured recombinanthost cells.

In addition, the present invention provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and at least one ofsuch an expression vector or recombinant virus comprising suchexpression vectors. The present invention further includespharmaceutical compositions, comprising a pharmaceutically acceptablecarrier and a polypeptide described herein.

The present invention also contemplates methods for detecting thepresence of Zcytor14 RNA in a biological sample, comprising the steps of(a) contacting a Zcytor14 nucleic acid probe under hybridizingconditions with either (i) test RNA molecules isolated from thebiological sample, or (ii) nucleic acid molecules synthesized from theisolated RNA molecules, wherein the probe has a nucleotide sequencecomprising a portion of the nucleotide sequence of SEQ ID NO:1, or itscomplement, and (b) detecting the formation of hybrids of the nucleicacid probe and either the test RNA molecules or the synthesized nucleicacid molecules, wherein the presence of the hybrids indicates thepresence of Zcytor14 RNA in the biological sample.

The present invention further provides methods for detecting thepresence of Zcytor14 polypeptide in a biological sample, comprising thesteps of: (a) contacting the biological sample with an antibody or anantibody fragment that specifically binds with a polypeptide consistingof the amino acid sequence of SEQ ID NO:2, wherein the contacting isperformed under conditions that allow the binding of the antibody orantibody fragment to the biological sample, and (b) detecting any of thebound antibody or bound antibody fragment. Such an antibody or antibodyfragment may further comprise a detectable label selected from the groupconsisting of radioisotope, fluorescent label, chemiluminescent label,enzyme label, bioluminescent label, and colloidal gold.

The present invention also provides kits for performing these detectionmethods. For example, a kit for detection of Zcytor14 gene expressionmay comprise a container that comprises a nucleic acid molecule, whereinthe nucleic acid molecule is selected from the group consisting of (a) anucleic acid molecule comprising the nucleotide sequence of nucleotides214 to 2184 of SEQ ID NO:1, (b) a nucleic acid molecule comprising thecomplement of nucleotides 214 to 2184 of the nucleotide sequence of SEQID NO:1, (c) a nucleic acid molecule that is a fragment of (a)consisting of at least eight nucleotides, and (d) a nucleic acidmolecule that is a fragment of (b) consisting of at least eightnucleotides. Such a kit may also comprise a second container thatcomprises one or more reagents capable of indicating the presence of thenucleic acid molecule. On the other hand, a kit for detection ofZcytor14 protein may comprise a container that comprises an antibody, oran antibody fragment, that specifically binds with a polypeptideconsisting of the amino acid sequence of SEQ ID NO:2.

The present invention also contemplates anti-idiotype antibodies, oranti-idiotype antibody fragments, that specifically bind an antibody orantibody fragment that specifically binds a polypeptide consisting ofthe amino acid sequence of SEQ ID NO:2 or SEQ ID NO:10.

The present invention also provides isolated nucleic acid moleculescomprising a nucleotide sequence that encodes a Zcytor14 secretionsignal sequence and a nucleotide sequence that encodes a biologicallyactive polypeptide, wherein the Zcytor14 secretion signal sequencecomprises an amino acid sequence of residues 1 to 20 of SEQ ID NO:2.Illustrative biologically active polypeptides include Factor VIIa,proinsulin, insulin, follicle stimulating hormone, tissue typeplasminogen activator, tumor necrosis factor, interleukin, colonystimulating factor, interferon, erythropoietin, and thrombopoietin.Moreover, the present invention provides fusion proteins comprising aZcytor14 secretion signal sequence and a polypeptide, wherein theZcytor14 secretion signal sequence comprises an amino acid sequence ofresidues 1 to 20 of SEQ ID NO:2.

The present invention further contemplates isolated nucleic acidmolecules that encode an extracellular Zcytor14 domain, wherein theextracellular domain comprises either amino acid residues 21 to 452 ofSEQ ID NO:2, or amino acid residues 21 to 435 of SEQ ID NO:10. Thepresent invention also includes isolated polypeptides consisting ofeither amino acid residues 21 to 452 of SEQ ID NO:2, or amino acidresidues 21 to 435 of SEQ ID NO:10, antibodies that specifically bindsuch polypeptides, and anti-idiotype antibodies that specifically bindwith such antibodies.

The present invention also provides fusion proteins, comprising aZcytor14 extracellular domain and an immunoglobulin moiety, wherein theZcytor14 extracellular domain comprises either amino acid residues 21 to452 of SEQ ID NO:2, or amino acid residues 21 to 435 of SEQ ID NO:10. Insuch fusion proteins, the immunoglobulin moiety may be an immunoglobulinheavy chain constant region, such as a human F_(c) fragment. The presentinvention further includes isolated nucleic acid molecules that encodesuch fusion proteins.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

2. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a nucleic acid molecule that has a contiguousstretch of identical or complementary sequence to another nucleic acidmolecule. Contiguous sequences are said to “overlap” a given stretch ofa nucleic acid molecule either in their entirety or along a partialstretch of the nucleic acid molecule.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner. For example, aZcytor14 promoter should stimulate expression of a operably linked geneto a greater extent in thyroid, adrenal gland, prostate, and livertissues, as opposed to kidney, pancreas, or spleen tissues.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentinvention, integrated genetic elements are most commonly derived fromlinearized plasmids that are introduced into the cells byelectroporation or other techniques. Integrated genetic elements arepassed from the original host cell to its progeny.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that producesZcytor14 from an expression vector. In contrast, Zcytor14 can beproduced by a cell that is a “natural source” of Zcytor14, and thatlacks an expression vector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a Zcytor14polypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities ofZcytor14 using affinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a polypeptide encoded by asplice variant of an mRNA transcribed from a gene.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

An “anti-idiotype antibody” is an antibody that binds with the variableregion domain of an immunoglobulin. In the present context, ananti-idiotype antibody binds with the variable region of ananti-Zcytor14 antibody, and thus, an anti-idiotype antibody mimics anepitope of Zcytor14.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-Zcytor14 monoclonal antibody fragmentbinds with an epitope of Zcytor14.

The term “antibody fragment” also includes a synthetic or a geneticallyengineered polypeptide that binds to a specific antigen, such aspolypeptides consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains and complementary determining regions derived from arodent antibody, while the remainder of the antibody molecule is derivedfrom a human antibody.

“Humanized antibodies” are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

As used herein, a “therapeutic agent” is a molecule or atom, which isconjugated to an antibody moiety to produce a conjugate, which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom, which can be conjugated toan antibody moiety to produce a molecule useful for diagnosis. Examplesof detectable labels include chelators, photoactive agents,radioisotopes, fluorescent agents, paramagnetic ions, or other markermoieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

A “naked antibody” is an entire antibody, as opposed to an antibodyfragment, which is not conjugated with a therapeutic agent. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric and humanizedantibodies.

As used herein, the term “antibody component” includes both an entireantibody and an antibody fragment.

An “immunoconjugate” is a conjugate of an antibody component with atherapeutic agent or a detectable label.

As used herein, the term “antibody fusion protein” refers to arecombinant molecule that comprises an antibody component and a Zcytor14polypeptide component. Examples of an antibody fusion protein include aprotein that comprises a Zcytor14 extracellular domain, and either an Fcdomain or an antigen-biding region.

A “target polypeptide” or a “target peptide” is an amino acid sequencethat comprises at least one epitope, and that is expressed on a targetcell, such as a tumor cell, or a cell that carries an infectious agentantigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

An “antigenic peptide” is a peptide that will bind a majorhistocompatibility complex molecule to form an MHC-peptide complex,which is recognized by a T cell, thereby inducing a cytotoxic lymphocyteresponse upon presentation to the T cell. Thus, antigenic peptides arecapable of binding to an appropriate major histocompatibility complexmolecule and inducing a cytotoxic T cells response, such as cell lysisor specific cytokine release against the target cell, which binds orexpresses the antigen. The antigenic peptide can be bound in the contextof a class I or class II major histocompatibility complex molecule, onan antigen presenting cell or on a target cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for Zcytor14” or a “Zcytor14anti-sense oligonucleotide” is an oligonucleotide having a sequence (a)capable of forming a stable triplex with a portion of the Zcytor14 gene,or (b) capable of forming a stable duplex with a portion of an mRNAtranscript of the Zcytor14 gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

An “external guide sequence” is a nucleic acid molecule that directs theendogenous ribozyme, RNase P, to a particular species of intracellularmRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acidmolecule that encodes an external guide sequence is termed an “externalguide sequence gene.”

The term “variant Zcytor14 gene” refers to nucleic acid molecules thatencode a polypeptide having an amino acid sequence that is amodification of SEQ ID NO:2. Such variants include naturally-occurringpolymorphisms of Zcytor14 genes, as well as synthetic genes that containconservative amino acid substitutions of the amino acid sequence of SEQID NO:2. Additional variant forms of Zcytor14 genes are nucleic acidmolecules that contain insertions or deletions of the nucleotidesequences described herein. A variant Zcytor14 gene can be identified,for example, by determining whether the gene hybridizes with a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:1, or itscomplement, under stringent conditions.

Alternatively, variant Zcytor14 genes can be identified by sequencecomparison. Two amino acid sequences have “100% amino acid sequenceidentity” if the amino acid residues of the two amino acid sequences arethe same when aligned for maximal correspondence. Similarly, twonucleotide sequences have “100% nucleotide sequence identity” if thenucleotide residues of the two nucleotide sequences are the same whenaligned for maximal correspondence. Sequence comparisons can beperformed using standard software programs such as those included in theLASERGENE bioinformatics computing suite, which is produced by DNASTAR(Madison, Wis.). Other methods for comparing two nucleotide or aminoacid sequences by determining optimal alignment are well-known to thoseof skill in the art (see, for example, Peruski and Peruski, The Internetand the New Biology: Tools for Genomic and Molecular Research (ASMPress, Inc. 1997), Wu et al. (eds.), “Information Superhighway andComputer Databases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.1998)). Particular methods for determining sequence identity aredescribed below.

Regardless of the particular method used to identify a variant Zcytor14gene or variant Zcytor14 polypeptide, a variant gene or polypeptideencoded by a variant gene may be functionally characterized the abilityto bind specifically to an anti-Zcytor14 antibody.

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

The present invention includes functional fragments of Zcytor14 genes.Within the context of this invention, a “functional fragment” of aZcytor14 gene refers to a nucleic acid molecule that encodes a portionof a Zcytor14 polypeptide, which is a domain described herein or atleast specifically binds with an anti-Zcytor14 antibody.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to ±10%.

3. Production of Zcytor14 Genes

Nucleic acid molecules encoding a human Zcytor14 gene can be obtained byscreening a human cDNA or genomic library using polynucleotide probesbased upon SEQ ID NO:1 or SEQ ID NO:4. These techniques are standard andwell-established.

As an illustration, a nucleic acid molecule that encodes a humanZcytor14 gene can be isolated from a cDNA library. In this case, thefirst step would be to prepare the cDNA library by isolating RNA from atissue, such as thyroid, adrenal gland, prostate, or liver tissues,using methods well-known to those of skill in the art. In general, RNAisolation techniques must provide a method for breaking cells, a meansof inhibiting RNase-directed degradation of RNA, and a method ofseparating RNA from DNA, protein, and polysaccharide contaminants. Forexample, total RNA can be isolated by freezing tissue in liquidnitrogen, grinding the frozen tissue with a mortar and pestle to lysethe cells, extracting the ground tissue with a solution ofphenol/chloroform to remove proteins, and separating RNA from theremaining impurities by selective precipitation with lithium chloride(see, for example, Ausubel et al. (eds.), Short Protocols in MolecularBiology, 3^(rd) Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995)[“Ausubel (1995)”]; Wu et al., Methods in Gene Biotechnology, pages33-41 (CRC Press, Inc. 1997) [“Wu (1997)”]).

Alternatively, total RNA can be isolated by extracting ground tissuewith guanidinium isothiocyanate, extracting with organic solvents, andseparating RNA from contaminants using differential centrifugation (see,for example, Chirgwin et al., Biochemistry 18:52 (1979); Ausubel (1995)at pages 4-1 to 4-6; Wu (1997) at pages 33-41).

In order to construct a cDNA library, poly(A)⁺ RNA must be isolated froma total RNA preparation. Poly(A)⁺ RNA can be isolated from total RNAusing the standard technique of oligo(dT)-cellulose chromatography (see,for example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972);Ausubel (1995) at pages 4-11 to 4-12).

Double-stranded cDNA molecules are synthesized from poly(A)⁺ RNA usingtechniques well-known to those in the art. (see, for example, Wu (1997)at pages 41-46). Moreover, commercially available kits can be used tosynthesize double-stranded cDNA molecules. For example, such kits areavailable from Life Technologies, Inc. (Gaithersburg, Md.), CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Promega Corporation (Madison,Wis.) and STRATAGENE (La Jolla, Calif.).

Various cloning vectors are appropriate for the construction of a cDNAlibrary. For example, a cDNA library can be prepared in a vector derivedfrom bacteriophage, such as a λgt10 vector. See, for example, Huynh etal., “Constructing and Screening cDNA Libraries in λgt10 and λgt11,” inDNA Cloning: A Practical Approach Vol. I, Glover (ed.), page 49 (IRLPress, 1985); Wu (1997) at pages 47-52.

Alternatively, double-stranded cDNA molecules can be inserted into aplasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla,Calif.), a LAMDAGEM-4 (Promega Corp.) or other commercially availablevectors. Suitable cloning vectors also can be obtained from the AmericanType Culture Collection (Manassas, Va.).

To amplify the cloned cDNA molecules, the cDNA library is inserted intoa prokaryotic host, using standard techniques. For example, a cDNAlibrary can be introduced into competent E. coli DH5 cells, which can beobtained, for example, from Life Technologies, Inc. (Gaithersburg, Md.).

A human genomic library can be prepared by means well-known in the art(see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) atpages 307-327). Genomic DNA can be isolated by lysing tissue with thedetergent Sarkosyl, digesting the lysate with proteinase K, clearinginsoluble debris from the lysate by centrifugation, precipitatingnucleic acid from the lysate using isopropanol, and purifyingresuspended DNA on a cesium chloride density gradient.

DNA fragments that are suitable for the production of a genomic librarycan be obtained by the random shearing of genomic DNA or by the partialdigestion of genomic DNA with restriction endonucleases. Genomic DNAfragments can be inserted into a vector, such as a bacteriophage orcosmid vector, in accordance with conventional techniques, such as theuse of restriction enzyme digestion to provide appropriate termini, theuse of alkaline phosphatase treatment to avoid undesirable joining ofDNA molecules, and ligation with appropriate ligases. Techniques forsuch manipulation are well-known in the art (see, for example, Ausubel(1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327).

Alternatively, human genomic libraries can be obtained from commercialsources such as Research Genetics (Huntsville, Ala.) and the AmericanType Culture Collection (Manassas, Va.).

A library containing cDNA or genomic clones can be screened with one ormore polynucleotide probes based upon SEQ ID NO:1 or SEQ ID NO:4, usingstandard methods (see, for example, Ausubel (1995) at pages 6-1 to6-11).

Nucleic acid molecules that encode a human Zcytor14 gene can also beobtained using the polymerase chain reaction (PCR) with oligonucleotideprimers having nucleotide sequences that are based upon the nucleotidesequences of the Zcytor14 gene, as described herein. General methods forscreening libraries with PCR are provided by, for example, Yu et al.,“Use of the Polymerase Chain Reaction to Screen Phage Libraries,” inMethods in Molecular Biology, Vol. 15: PCR Protocols: Current Methodsand Applications, White (ed.), pages 211-215 (Humana Press, Inc. 1993).Moreover, techniques for using PCR to isolate related genes aredescribed by, for example, Preston, “Use of Degenerate OligonucleotidePrimers and the Polymerase Chain Reaction to Clone Gene Family Members,”in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methodsand Applications, White (ed.), pages 317-337 (Humana Press, Inc. 1993).

Anti-Zcytor14 antibodies, produced as described below, can also be usedto isolate DNA sequences that encode human Zcytor14 genes from cDNAlibraries. For example, the antibodies can be used to screen λgt11expression libraries, or the antibodies can be used for immunoscreeningfollowing hybrid selection and translation (see, for example, Ausubel(1995) at pages 6-12 to 6-16; Margolis et al., “Screening λ expressionlibraries with antibody and protein probes,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), pages 1-14(Oxford University Press 1995)).

As an alternative, a Zcytor14 gene can be obtained by synthesizingnucleic acid molecules using mutually priming long oligonucleotides andthe nucleotide sequences described herein (see, for example, Ausubel(1995) at pages 8-8 to 8-9). Established techniques using the polymerasechain reaction provide the ability to synthesize DNA molecules at leasttwo kilobases in length (Adang et al., Plant Molec. Biol. 21:1131(1993), Bambot et al., PCR Methods and Applications 2:266 (1993), Dillonet al., “Use of the Polymerase Chain Reaction for the Rapid Constructionof Synthetic Genes,” in Methods in Molecular Biology, Vol. 15: PCRProtocols: Current Methods and Applications, White (ed.), pages 263-268,(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.4:299 (1995)).

The nucleic acid molecules of the present invention can also besynthesized with “gene machines” using protocols such as thephosphoramidite method. If chemically-synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 base pairs) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. For the production oflonger genes (>300 base pairs), however, special strategies may berequired, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length. For reviews onpolynucleotide synthesis, see, for example, Glick and Pasternak,Molecular Biotechnology, Principles and Applications of Recombinant DNA(ASM Press 1994), Itakura et al., Annu. Rev. Biochem. 53:323 (1984), andClimie et al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).

The sequence of a Zcytor14 cDNA or Zcytor14 genomic fragment can bedetermined using standard methods. Zcytor14 polynucleotide sequencesdisclosed herein can also be used as probes or primers to clone 5′non-coding regions of a Zcytor14 gene. Promoter elements from a Zcytor14gene can be used to direct the expression of heterologous genes in, forexample, thyroid tissue of transgenic animals or patients treated withgene therapy. The identification of genomic fragments containing aZcytor14 promoter or regulatory element can be achieved usingwell-established techniques, such as deletion analysis (see, generally,Ausubel (1995)).

Cloning of 5′ flanking sequences also facilitates production of Zcytor14proteins by “gene activation,” as disclosed in U.S. Pat. No. 5,641,670.Briefly, expression of an endogenous Zcytor14 gene in a cell is alteredby introducing into the Zcytor14 locus a DNA construct comprising atleast a targeting sequence, a regulatory sequence, an exon, and anunpaired splice donor site. The targeting sequence is a Zcytor14 5′non-coding sequence that permits homologous recombination of theconstruct with the endogenous Zcytor14 locus, whereby the sequenceswithin the construct become operably linked with the endogenous Zcytor14coding sequence. In this way, an endogenous Zcytor14 promoter can bereplaced or supplemented with other regulatory sequences to provideenhanced, tissue-specific, or otherwise regulated expression.

4. Production of Zcytor14 Gene Variants

The present invention provides a variety of nucleic acid molecules,including DNA and RNA molecules, that encode the Zcytor14 polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NO:3is a degenerate nucleotide sequence that encompasses all nucleic acidmolecules that encode the Zcytor14 polypeptide of SEQ ID NO:2. Thoseskilled in the art will recognize that the degenerate sequence of SEQ IDNO:3 also provides all RNA sequences encoding SEQ ID NO:2, bysubstituting U for T. Thus, the present invention contemplates Zcytor14polypeptide-encoding nucleic acid molecules comprising nucleotide 154 tonucleotide 2229 of SEQ ID NO:1, and their RNA equivalents. Similarly,the Zcytor14-1 degenerate sequence of SEQ ID NO:6 also provides all RNAsequences encoding SEQ ID NO:5, by substituting U for T.

Table 1 sets forth the one-letter codes used within SEQ ID NOs:3 and 6to denote degenerate nucleotide positions. “Resolutions” are thenucleotides denoted by a code letter. “Complement” indicates the codefor the complementary nucleotide(s). For example, the code Y denoteseither C or T, and its complement R denotes A or G, A beingcomplementary to T, and G being complementary to C. TABLE 1 NucleotideResolution Complement Resolution A A T T C C G G G G C C T T A A R A|G YC|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T W A|T H A|C|TD A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T NA|C|G|T

The degenerate codons used in SEQ ID NOs:3 and 6, encompassing allpossible codons for a given amino acid, are set forth in Table 2. TABLE2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGT TGYSer S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCACCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN AsnN AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR HisH CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met MATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val VGTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGGTer · TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding an amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequences of SEQ ID NOs:2, 5, and 10 to 12. Variant sequences can bereadily tested for functionality as described herein.

Different species can exhibit “preferential codon usage.” In general,see, Grantham et al., Nucl. Acids Res. 8:1893 (1980), Haas et al. Curr.Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355 (1981), Grosjean andFiers, Gene 18:199 (1982), Holm, Nuc. Acids Res. 14:3075 (1986),Ikemura, J. Mol. Biol. 158:573 (1982), Sharp and Matassi, Curr. Opin.Genet. Dev. 4:851 (1994), Kane, Curr. Opin. Biotechnol. 6:494 (1995),and Makrides, Microbiol. Rev. 60:512 (1996). As used herein, the term“preferential codon usage” or “preferential codons” is a term of artreferring to protein translation codons that are most frequently used incells of a certain species, thus favoring one or a few representativesof the possible codons encoding each amino acid (See Table 2). Forexample, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG,or ACT, but in mammalian cells ACC is the most commonly used codon; inother species, for example, insect cells, yeast, viruses or bacteria,different Thr codons may be preferential. Preferential codons for aparticular species can be introduced into the polynucleotides of thepresent invention by a variety of methods known in the art. Introductionof preferential codon sequences into recombinant DNA can, for example,enhance production of the protein by making protein translation moreefficient within a particular cell type or species. Therefore, thedegenerate codon sequences disclosed herein serve as a template foroptimizing expression of polynucleotides in various cell types andspecies commonly used in the art and disclosed herein. Sequencescontaining preferential codons can be tested and optimized forexpression in various species, and tested for functionality as disclosedherein.

The present invention further provides variant polypeptides and nucleicacid molecules that represent counterparts from other species(orthologs). These species include, but are not limited to mammalian,avian, amphibian, reptile, fish, insect and other vertebrate andinvertebrate species. Of particular interest are Zcytor14 polypeptidesfrom other mammalian species, including mouse, porcine, ovine, bovine,canine, feline, equine, and other primate polypeptides. Orthologs ofhuman Zcytor14 can be cloned using information and compositions providedby the present invention in combination with conventional cloningtechniques. For example, a Zcytor14 cDNA can be cloned using mRNAobtained from a tissue or cell type that expresses Zcytor14 as disclosedherein. Suitable sources of mRNA can be identified by probing northernblots with probes designed from the sequences disclosed herein. Alibrary is then prepared from mRNA of a positive tissue or cell line.

A Zcytor14-encoding cDNA can be isolated by a variety of methods, suchas by probing with a complete or partial human cDNA or with one or moresets of degenerate probes based on the disclosed sequences. A cDNA canalso be cloned using the polymerase chain reaction with primers designedfrom the representative human Zcytor14 sequences disclosed herein. Inaddition, a cDNA library can be used to transform or transfect hostcells, and expression of the cDNA of interest can be detected with anantibody to Zcytor14 polypeptide.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of human Zcytor14, and thatallelic variation and alternative splicing are expected to occur.Allelic variants of this sequence can be cloned by probing cDNA orgenomic libraries from different individuals according to standardprocedures. Allelic variants of the nucleotide sequences disclosedherein, including those containing silent mutations and those in whichmutations result in amino acid sequence changes, are within the scope ofthe present invention, as are proteins, which are allelic variants ofthe amino acid sequences disclosed herein. cDNA molecules generated fromalternatively spliced mRNAs, which retain the properties of the Zcytor14polypeptide are included within the scope of the present invention, asare polypeptides encoded by such cDNAs and mRNAs. Allelic variants andsplice variants of these sequences can be cloned by probing cDNA orgenomic libraries from different individuals or tissues according tostandard procedures known in the art.

Within certain embodiments of the invention, the isolated nucleic acidmolecules can hybridize under stringent conditions to nucleic acidmolecules comprising nucleotide sequences disclosed herein. For example,such nucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules comprising the nucleotide sequence of SEQ IDNO:1, to nucleic acid molecules consisting of the nucleotide sequence ofnucleotides 154 to 2229 of SEQ ID NO:1, or to nucleic acid moleculescomprising a nucleotide sequence complementary to SEQ ID NO:1 or tonucleotides 154 to 2229 of SEQ ID NO:1. In general, stringent conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases. Stringent hybridization conditionsencompass temperatures of about 5-25° C. below the T_(m) of the hybridand a hybridization buffer having up to 1 M Na⁺. Higher degrees ofstringency at lower temperatures can be achieved with the addition offormamide, which reduces the T_(m) of the hybrid about 1° C. for each 1%formamide in the buffer solution. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide. A higher degree of stringency can beachieved at temperatures of from 40-70° C. with a hybridization bufferhaving up to 4×SSC and from 0-50% formamide. Highly stringent conditionstypically encompass temperatures of 42-70° C. with a hybridizationbuffer having up to 1×SSC and 0-50% formamide. Different degrees ofstringency can be used during hybridization and washing to achievemaximum specific binding to the target sequence. Typically, the washesfollowing hybridization are performed at increasing degrees ofstringency to remove non-hybridized polynucleotide probes fromhybridized complexes.

The above conditions are meant to serve as a guide and it is well withinthe abilities of one skilled in the art to adapt these conditions foruse with a particular polypeptide hybrid. The T_(m) for a specifictarget sequence is the temperature (under defined conditions) at which50% of the target sequence will hybridize to a perfectly matched probesequence. Those conditions that influence the T_(m) include, the sizeand base pair content of the polynucleotide probe, the ionic strength ofthe hybridization solution, and the presence of destabilizing agents inthe hybridization solution. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake,Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto,Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating T_(m) based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences, >50 base pairs, isperformed at temperatures of about 20-25° C. below the calculated T_(m),For smaller probes, <50 base pairs, hybridization is typically carriedout at the T_(m) or 5-10° C. below. This allows for the maximum rate ofhybridization for DNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence influences the rate andstability of hybrid formation. Smaller probe sequences, <50 base pairs,reach equilibrium with complementary sequences rapidly, but may formless stable hybrids. Incubation times of anywhere from minutes to hourscan be used to achieve hybrid formation. Longer probe sequences come toequilibrium more slowly, but form more stable complexes even at lowertemperatures. Incubations are allowed to proceed overnight or longer.Generally, incubations are carried out for a period equal to three timesthe calculated Cot time. Cot time, the time it takes for thepolynucleotide sequences to reassociate, can be calculated for aparticular sequence by methods known in the art.

The base pair composition of polynucleotide sequence will effect thethermal stability of the hybrid complex, thereby influencing the choiceof hybridization temperature and the ionic strength of the hybridizationbuffer. A-T pairs are less stable than G-C pairs in aqueous solutionscontaining sodium chloride. Therefore, the higher the G-C content, themore stable the hybrid. Even distribution of G and C residues within thesequence also contribute positively to hybrid stability. In addition,the base pair composition can be manipulated to alter the T_(m) of agiven sequence. For example, 5-methyldeoxycytidine can be substitutedfor deoxycytidine and 5-bromodeoxuridine can be substituted forthymidine to increase the T_(m), whereas 7-deazz-2′-deoxyguanosine canbe substituted for guanosine to reduce dependence on T_(m).

The ionic concentration of the hybridization buffer also affects thestability of the hybrid. Hybridization buffers generally containblocking agents such as Denhardt's solution (Sigma Chemical Co., St.Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO),heparin or SDS, and a Na⁺ source, such as SSC (1×SSC: 0.15 M sodiumchloride, 15 mM sodium citrate) or SSPE (1×SSPE: 1.8 M NaCl, 10 mMNaH₂PO₄, 1 mM EDTA, pH 7.7). Typically, hybridization buffers containfrom between 10 mM -1 M Na⁺. The addition of destabilizing or denaturingagents such as formamide, tetralkylammonium salts, guanidinium cationsor thiocyanate cations to the hybridization solution will alter theT_(m) of a hybrid. Typically, formamide is used at a concentration of upto 50% to allow incubations to be carried out at more convenient andlower temperatures. Formamide also acts to reduce non-specificbackground when using RNA probes.

As an illustration, a nucleic acid molecule encoding a variant Zcytor14polypeptide can be hybridized with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) at 42° C.overnight in a solution comprising 50% formamide, 5×SSC, 50 mM sodiumphosphate (pH 7.6), 5× Denhardt's solution (100× Denhardt's solution: 2%(w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovineserum albumin), 10% dextran sulfate, and 20 μg/ml denatured, shearedsalmon sperm DNA. One of skill in the art can devise variations of thesehybridization conditions. For example, the hybridization mixture can beincubated at a higher temperature, such as about 65° C., in a solutionthat does not contain formamide. Moreover, premixed hybridizationsolutions are available (e.g., EXPRESSHYB Hybridization Solution fromCLONTECH Laboratories, Inc.), and hybridization can be performedaccording to the manufacturer's instructions.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. As an illustration, nucleic acidmolecules encoding a variant Zcytor14 polypeptide remain hybridized witha nucleic acid molecule consisting of the nucleotide sequence of SEQ IDNO:1 (or its complement) following stringent washing conditions, inwhich the wash stringency is equivalent to 0.5×-2×SSC with 0.1% SDS at55-65° C., including 0.5×SSC with 0.1% SDS at 55° C., or 2×SSC with 0.1%SDS at 65° C. One of skill in the art can readily devise equivalentconditions, for example, by substituting SSPE for SSC in the washsolution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. For example, nucleic acid molecules encoding a variantZcytor14 polypeptide remain hybridized with a nucleic acid moleculeconsisting of the nucleotide sequence of SEQ ID NO:1 (or its complement)following highly stringent washing conditions, in which the washstringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C.,including 0.1×SSC with 0.1% SDS at 50° C., or 0.2×SSC with 0.1% SDS at65° C.

The present invention also provides isolated Zcytor14 polypeptides thathave a substantially similar sequence identity to the polypeptides ofSEQ ID NO:2, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides having at least70%, at least 80%, at least 90%, at least 95% or greater than 95%sequence identity to the sequences shown in SEQ ID NO:2, or theirorthologs.

The present invention also contemplates Zcytor14 variant nucleic acidmolecules that can be identified using two criteria: a determination ofthe similarity between the encoded polypeptide with the amino acidsequence of SEQ ID NO:2, and a hybridization assay, as described above.Such Zcytor14 variants include nucleic acid molecules (1) that remainhybridized with a nucleic acid molecule consisting of the nucleotidesequence of SEQ ID NO:1 (or its complement) following stringent washingconditions, in which the wash stringency is equivalent to 0.5×-2×SSCwith 0.1% SDS at 55-65° C., and (2) that encode a polypeptide having atleast 70%, at least 80%, at least 90%, at least 95% or greater than 95%sequence identity to the amino acid sequence of SEQ ID NO:2.Alternatively, Zcytor14 variants can be characterized as nucleic acidmolecules (1) that remain hybridized with a nucleic acid moleculeconsisting of the nucleotide sequence of SEQ ID NO:1 (or its complement)following highly stringent washing conditions, in which the washstringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., and(2) that encode a polypeptide having at least 70%, at least 80%, atleast 90%, at least 95% or greater than 95% sequence identity to theamino acid sequence of SEQ ID NO:2.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as: ([Total number ofidentical matches]/[length of the longer sequence plus the number ofgaps introduced into the longer sequence in order to align the twosequences])(100). TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R−1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 25 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3−4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −25 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0−3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0−1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W−3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1−2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1−1 −2 −2 0 −3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativeZcytor14 variant. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequencesimilarity by identifying regions shared by the query sequence (e.g.,SEQ ID NO:2) and a test sequence that have either the highest density ofidentities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdescribed above.

The present invention includes nucleic acid molecules that encode apolypeptide having a conservative amino acid change, compared with anamino acid sequence disclosed herein. For example, variants can beobtained that contain one or more amino acid substitutions of SEQ IDNO:2, in which an alkyl amino acid is substituted for an alkyl aminoacid in a Zcytor14 amino acid sequence, an aromatic amino acid issubstituted for an aromatic amino acid in a Zcytor14 amino acidsequence, a sulfur-containing amino acid is substituted for asulfur-containing amino acid in a Zcytor14 amino acid sequence, ahydroxy-containing amino acid is substituted for a hydroxy-containingamino acid in a Zcytor14 amino acid sequence, an acidic amino acid issubstituted for an acidic amino acid in a Zcytor14 amino acid sequence,a basic amino acid is substituted for a basic amino acid in a Zcytor14amino acid sequence, or a dibasic monocarboxylic amino acid issubstituted for a dibasic monocarboxylic amino acid in a Zcytor14 aminoacid sequence. Among the common amino acids, for example, a“conservative amino acid substitution” is illustrated by a substitutionamong amino acids within each of the following groups: (1) glycine,alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine,and tryptophan, (3) serine and threonine, (4) aspartate and glutamate,(5) glutamine and asparagine, and (6) lysine, arginine and histidine.

The BLOSUM62 table is an amino acid substitution matrix derived fromabout 2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915(1992)). Accordingly, the BLOSUM62 substitution frequencies can be usedto define conservative amino acid substitutions that may be introducedinto the amino acid sequences of the present invention. Although it ispossible to design amino acid substitutions based solely upon chemicalproperties (as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

Particular variants of Zcytor14 are characterized by having at least70%, at least 80%, at least 90%, at least 95% or greater than 95%sequence identity to the corresponding amino acid sequence (e.g., SEQ IDNO:2), wherein the variation in amino acid sequence is due to one ormore conservative amino acid substitutions.

Conservative amino acid changes in a Zcytor14 gene can be introduced,for example, by substituting nucleotides for the nucleotides recited inSEQ ID NO:1. Such “conservative amino acid” variants can be obtained byoligonucleotide-directed mutagenesis, linker-scanning mutagenesis,mutagenesis using the polymerase chain reaction, and the like (seeAusubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), DirectedMutagenesis: A Practical Approach (IRL Press 1991)). A variant Zcytor14polypeptide can be identified by the ability to specifically bindanti-Zcytor14 antibodies.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is typicallycarried out in a cell-free system comprising an E. coli S30 extract andcommercially available enzymes and other reagents. Proteins are purifiedby chromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993)).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for Zcytor14 amino acidresidues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Nat'l Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., J. Biol. Chem.271:4699 (1996).

Although sequence analysis can be used to further define the Zcytor14ligand binding region, amino acids that play a role in Zcytor14 bindingactivity can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., Science 255:306 (1992), Smith et al., J. Mol.Biol. 224:899(1992), and Wlodaveret al., FEBS Lett. 309:59 (1992).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204, and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)). Moreover, Zcytor14labeled with biotin or FITC can be used for expression cloning.

Variants of the disclosed Zcytor14 nucleotide and polypeptide sequencescan also be generated through DNA shuffling as disclosed by Stemmer,Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci. USA 91:10747(1994), and international publication No. WO 97/20078. Briefly, variantDNA molecules are generated by in vitro homologous recombination byrandom fragmentation of a parent DNA followed by reassembly using PCR,resulting in randomly introduced point mutations. This technique can bemodified by using a family of parent DNA molecules, such as allelicvariants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-Zcytor14 antibodies, can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

The present invention also includes “functional fragments” of Zcytor14polypeptides and nucleic acid molecules encoding such functionalfragments. Routine deletion analyses of nucleic acid molecules can beperformed to obtain functional fragments of a nucleic acid molecule thatencodes a Zcytor14 polypeptide. As an illustration, DNA molecules havingthe nucleotide sequence of SEQ ID NO:1 can be digested with Bal31nuclease to obtain a series of nested deletions. The fragments are theninserted into expression vectors in proper reading frame, and theexpressed polypeptides are isolated and tested for the ability to bindanti-Zcytor14 antibodies. One alternative to exonuclease digestion is touse oligonucleotide-directed mutagenesis to introduce deletions or stopcodons to specify production of a desired fragment. Alternatively,particular fragments of a Zcytor14 gene can be synthesized using thepolymerase chain reaction.

As an illustration of this general approach, studies on the truncationat either or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993),Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987), Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation, Vol. 1, Boynton etal., (eds.) pages 169-199 (Academic Press 1985), Coumailleau et al., J.Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995), and Meiselet al., Plant Molec. Biol. 30:1 (1996).

An example of a functional fragment of a Zcytor14 polypeptide is asoluble form of Zcytor14 that lacks a transmembrane domain. IllustrativeZcytor14 soluble forms include polypeptides consisting of amino acidresidues 1 to 452 of SEQ ID NO:2, amino acid residues 21 to 452 of SEQID NO:2, amino acid residues 1 to 435 of SEQ ID NO:10, or amino acidresidues 21 to 435 of SEQ ID NO:10.

The present invention also contemplates functional fragments of aZcytor14 gene that have amino acid changes, compared with an amino acidsequence disclosed herein. A variant Zcytor14 gene can be identified onthe basis of structure by determining the level of identity withdisclosed nucleotide and amino acid sequences, as discussed above. Analternative approach to identifying a variant gene on the basis ofstructure is to determine whether a nucleic acid molecule encoding apotential variant Zcytor14 gene can hybridize to a nucleic acid moleculecomprising a nucleotide sequence, such as SEQ ID NO:1 or SEQ ID NO:4.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a Zcytor14 polypeptidedescribed herein. Such fragments or peptides may comprise an“immunogenic epitope,” which is a part of a protein that elicits anantibody response when the entire protein is used as an immunogen.Immunogenic epitope-bearing peptides can be identified using standardmethods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodiesthat bind with the polypeptides described herein.

Antigenic epitope-bearing peptides and polypeptides can contain at leastfour to ten amino acids, at least ten to fifteen amino acids, or about15 to about 30 amino acids of an amino acid sequence disclosed herein.Such epitope-bearing peptides and polypeptides can be produced byfragmenting a Zcytor14 polypeptide, or by chemical peptide synthesis, asdescribed herein. Moreover, epitopes can be selected by phage display ofrandom peptide libraries (see, for example, Lane and Stephen, Curr.Opin. Immunol. 5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol.7:616 (1996)). Standard methods for identifying epitopes and producingantibodies from small peptides that comprise an epitope are described,for example, by Mole, “Epitope Mapping,” in Methods in MolecularBiology, Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc.1992), Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages60-84 (Cambridge University Press 1995), and Coligan et al. (eds.),Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997).

For any Zcytor14 polypeptide, including variants and fusion proteins,one of ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 1 and 2 above. Moreover, those of skill in the art canuse standard software to devise Zcytor14 variants based upon thenucleotide and amino acid sequences described herein. Accordingly, thepresent invention includes a computer-readable medium encoded with adata structure that provides at least one of the following sequences:SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ IDNO:12. Suitable forms of computer-readable media include magnetic mediaand optically-readable media. Examples of magnetic media include a hardor fixed drive, a random access memory (RAM) chip, a floppy disk,digital linear tape (DLT), a disk cache, and a ZIP disk. Opticallyreadable media are exemplified by compact discs (e.g., CD-read onlymemory (ROM), CD-rewritable (RW), and CD-recordable), and digitalversatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).

6. Production of Zcytor14 Polypeptides

The polypeptides of the present invention, including full-lengthpolypeptides, functional fragments, and fusion proteins, can be producedin recombinant host cells following conventional techniques. To expressa Zcytor14 gene, a nucleic acid molecule encoding the polypeptide mustbe operably linked to regulatory sequences that control transcriptionalexpression in an expression vector and then, introduced into a hostcell. In addition to transcriptional regulatory sequences, such aspromoters and enhancers, expression vectors can include translationalregulatory sequences and a marker gene, which is suitable for selectionof cells that carry the expression vector.

Expression vectors that are suitable for production of a foreign proteinin eukaryotic cells typically contain (1) prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancemarker to provide for the growth and selection of the expression vectorin a bacterial host; (2) eukaryotic DNA elements that control initiationof transcription, such as a promoter; and (3) DNA elements that controlthe processing of transcripts, such as a transcriptiontermination/polyadenylation sequence. As discussed above, expressionvectors can also include nucleotide sequences encoding a secretorysequence that directs the heterologous polypeptide into the secretorypathway of a host cell. For example, a Zcytor14 expression vector maycomprise a Zcytor14 gene and a secretory sequence derived from anysecreted gene.

Zcytor14 proteins of the present invention may be expressed in mammaliancells. Examples of suitable mammalian host cells include African greenmonkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells(293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570;ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34),Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin etal., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1;ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC CRL1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, in which the regulatorysignals are associated with a particular gene, which has a high level ofexpression. Suitable transcriptional and translational regulatorysequences also can be obtained from mammalian genes, such as actin,collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 earlypromoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma viruspromoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), thecytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control Zcytor14 gene expression inmammalian cells if the prokaryotic promoter is regulated by a eukaryoticpromoter (Zhou et al., Mol. Cell. Biol. 10:4529 (1990), and Kaufman etal., Nucl. Acids Res. 19:4485 (1991)).

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. The transfected cells can be selected andpropagated to provide recombinant host cells that comprise theexpression vector stably integrated in the host cell genome. Techniquesfor introducing vectors into eukaryotic cells and techniques forselecting such stable transformants using a dominant selectable markerare described, for example, by Ausubel (1995) and by Murray (ed.), GeneTransfer and Expression Protocols (Humana Press 1991).

For example, one suitable selectable marker is a gene that providesresistance to the antibiotic neomycin. In this case, selection iscarried out in the presence of a neomycin-type drug, such as G-418 orthe like. Selection systems can also be used to increase the expressionlevel of the gene of interest, a process referred to as “amplification.”Amplification is carried out by culturing transfectants in the presenceof a low level of the selective agent and then increasing the amount ofselective agent to select for cells that produce high levels of theproducts of the introduced genes. A suitable amplifiable selectablemarker is dihydrofolate reductase, which confers resistance tomethotrexate. Other drug resistance genes (e.g., hygromycin resistance,multi-drug resistance, puromycin acetyltransferase) can also be used.Alternatively, markers that introduce an altered phenotype, such asgreen fluorescent protein, or cell surface proteins such as CD4, CD8,Class I MHC, placental alkaline phosphatase may be used to sorttransfected cells from untransfected cells by such means as FACS sortingor magnetic bead separation technology.

Zcytor14 polypeptides can also be produced by cultured mammalian cellsusing a viral delivery system. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, vaccinia virus and adeno-associatedvirus (AAV). Adenovirus, a double-stranded DNA virus, is currently thebest studied gene transfer vector for delivery of heterologous nucleicacid (for a review, see Becker et al., Meth. Cell Biol. 43:161 (1994),and Douglas and Curiel, Science & Medicine 4:44 (1997)). Advantages ofthe adenovirus system include the accommodation of relatively large DNAinserts, the ability to grow to high-titer, the ability to infect abroad range of mammalian cell types, and flexibility that allows usewith a large number of available vectors containing different promoters.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. An option is to delete theessential E1 gene from the viral vector, which results in the inabilityto replicate unless the E1 gene is provided by the host cell. Adenovirusvector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), forexample, can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(see Gamier et al., Cytotechnol. 15:145 (1994)).

Zcytor14 can also be expressed in other higher eukaryotic cells, such asavian, fungal, insect, yeast, or plant cells. The baculovirus systemprovides an efficient means to introduce cloned Zcytor14 genes intoinsect cells. Suitable expression vectors are based upon the Autographacalifornica multiple nuclear polyhedrosis virus (AcMNPV), and containwell-known promoters such as Drosophila heat shock protein (hsp) 70promoter, Autographa californica nuclear polyhedrosis virusimmediate-early gene promoter (ie-1) and the delayed early 39K promoter,baculovirus p10 promoter, and the Drosophila metallothionein promoter. Asecond method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566 (1993)). This system, which utilizes transfer vectors, is soldin the BAC-to-BAC kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, PFASTBAC (Life Technologies) containing aTn7 transposon to move the DNA encoding the Zcytor14 polypeptide into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990),Bonning, et al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk, andRapoport, J. Biol. Chem. 270:1543 (1995). In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed Zcytor14 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer et al., Proc. Nat'l Acad. Sci. 82:7952(1985)). Using a technique known in the art, a transfer vectorcontaining a Zcytor14 gene is transformed into E. coli, and screened forbacmids, which contain an interrupted lacZ gene indicative ofrecombinant baculovirus. The bacmid DNA containing the recombinantbaculovirus genome is then isolated using common techniques.

The illustrative PFASTBAC vector can be modified to a considerabledegree. For example, the polyhedrin promoter can be removed andsubstituted with the baculovirus basic protein promoter (also known asPcor, p6.9 or MP promoter), which is expressed earlier in thebaculovirus infection, and has been shown to be advantageous forexpressing secreted proteins (see, for example, Hill-Perkins and Possee,J. Gen. Virol. 71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551(1994), and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). Insuch transfer vector constructs, a short or long version of the basicprotein promoter can be used. Moreover, transfer vectors can beconstructed, which replace the native Zcytor14 secretory signalsequences with secretory signal sequences derived from insect proteins.For example, a secretory signal sequence from EcdysteroidGlucosyltransferase (EGT), honey bee Melittin (Invitrogen Corporation;Carlsbad, Calif.), or baculovirus gp67 (PharMingen: San Diego, Calif.)can be used in constructs to replace the native Zcytor14 secretorysignal sequence.

The recombinant virus or bacmid is used to transfect host cells.Suitable insect host cells include cell lines derived from IPLB-Sf-21, aSpodoptera frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL1711), Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, Calif.), aswell as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line(Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).Commercially available serum-free media can be used to grow and tomaintain the cells. Suitable media are Sf900 II™ (Life Technologies) orESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. When recombinant virus is used, the cells are typicallygrown up from an inoculation density of approximately 2-5×10⁵ cells to adensity of 1-2×10⁶ cells at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3.

Established techniques for producing recombinant proteins in baculovirussystems are provided by Bailey et al., “Manipulation of BaculovirusVectors,” in Methods in Molecular Biology, Volume 7: Gene Transfer andExpression Protocols, Murray (ed.), pages 147-168 (The Humana Press,Inc. 1991), by Patel et al., “The baculovirus expression system,” in DNACloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995), and by Lucknow, “Insect Cell ExpressionTechnology,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).

Fungal cells, including yeast cells, can also be used to express thegenes described herein. Yeast species of particular interest in thisregard include Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Suitable promoters for expression in yeast includepromoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH(alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. Many yeast cloning vectors have beendesigned and are readily available. These vectors include YIp-basedvectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such asYEp13 and YCp vectors, such as YCp19. Methods for transforming S.cerevisiae cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake,U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A suitable vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Additional suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311, Kingsman etal., U.S. Pat. No. 4,615,974, and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446,5,063,154, 5,139,936, and 4,661,454.

Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459 (1986), and Cregg,U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according tothe methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods fortransforming Acremonium chrysogenum are disclosed by Sumino et al., U.S.Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533.

For example, the use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed by Raymond, U.S. Pat. No. 5,716,808,Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast 14:11-23 (1998),and in international publication Nos. WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A suitable selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), andwhich allows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, host cells can be used in which both methanolutilization genes (AUG1 and AUG2) are deleted. For production ofsecreted proteins, host cells deficient in vacuolar protease genes (PEP4and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. P. methanolica cells can betransformed by electroporation using an exponentially decaying, pulsedelectric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Expression vectors can also be introduced into plant protoplasts, intactplant tissues, or isolated plant cells. Methods for introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant tissue with Agrobacterium tumefaciens,microprojectile-mediated delivery, DNA injection, electroporation, andthe like. See, for example, Horsch et al., Science 227:1229 (1985),Klein et al., Biotechnology 10:268 (1992), and Miki et al., “Proceduresfor Introducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,1993).

Alternatively, Zcytor14 genes can be expressed in prokaryotic hostcells. Suitable promoters that can be used to express Zcytor14polypeptides in a prokaryotic host are well-known to those of skill inthe art and include promoters capable of recognizing the T4, T3, Sp6 andT7 polymerases, the P_(R) and P_(L) promoters of bacteriophage lambda,the trp, recA, heat shock, lacUV5, tac, lpp-lacSpr, phoA, and lacZpromoters of E. coli, promoters of B. subtilis, the promoters of thebacteriophages of Bacillus, Streptomyces promoters, the int promoter ofbacteriophage lambda, the bla promoter of pBR322, and the CAT promoterof the chloramphenicol acetyl transferase gene. Prokaryotic promotershave been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson etal., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), andby Ausubel et al. (1995).

Illustrative prokaryotic hosts include E. coli and Bacillus subtilus.Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF′, DH5IMCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, MI119, MI120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (ed.) (IRL Press 1985)).

When expressing a Zcytor14 polypeptide in bacteria such as E. coli, thepolypeptide may be retained in the cytoplasm, typically as insolublegranules, or may be directed to the periplasmic space by a bacterialsecretion sequence. In the former case, the cells are lysed, and thegranules are recovered and denatured using, for example, guanidineisothiocyanate or urea. The denatured polypeptide can then be refoldedand dimerized by diluting the denaturant, such as by dialysis against asolution of urea and a combination of reduced and oxidized glutathione,followed by dialysis against a buffered saline solution. In the lattercase, the polypeptide can be recovered from the periplasmic space in asoluble and functional form by disrupting the cells (by, for example,sonication or osmotic shock) to release the contents of the periplasmicspace and recovering the protein, thereby obviating the need fordenaturation and refolding.

Methods for expressing proteins in prokaryotic hosts are well-known tothose of skill in the art (see, for example, Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995), Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

Standard methods for introducing expression vectors into bacterial,yeast, insect, and plant cells are provided, for example, by Ausubel(1995).

General methods for expressing and recovering foreign protein producedby a mammalian cell system are provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example,Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.(eds.), pages 59-92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995).

As an alternative, polypeptides of the present invention can besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thesesynthesis methods are well-known to those of skill in the art (see, forexample, Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al.,“Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co.1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989),Fields and Colowick, “Solid-Phase Peptide Synthesis,” Methods inEnzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al.,Chemical Approaches to the Synthesis of Peptides and Proteins (CRCPress, Inc. 1997)). Variations in total chemical synthesis strategies,such as “native chemical ligation” and “expressed protein ligation” arealso standard (see, for example, Dawson et al., Science 266:776 (1994),Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205(1998)).

Peptides and polypeptides of the present invention comprise at leastsix, at least nine, or at least 15 contiguous amino acid residues of SEQID NO:2, SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. Forexample, the present invention includes polypeptides comprising, orconsisting of, 15 contiguous amino acids of the following amino acidsequences: amino acid residues 21 to 452 of the amino acid sequence ofSEQ ID NO:2, amino acid residues 21 to 435 of the amino acid sequence ofSEQ ID NO:10, amino acid residues 474 to 677 of SEQ ID NO:2, or aminoacid residues 457 to 673 of SEQ ID NO:10. Within certain embodiments ofthe invention, the polypeptides comprise 20, 30, 40, 50, 100, or morecontiguous residues of these amino acid sequences. As an illustration,the present invention includes polypeptides comprising, or consistingof, 30 or 40 contiguous amino acids of the following amino acidsequences: amino acid residues 21 to 452 of the amino acid sequence ofSEQ ID NO:2, amino acid residues 21 to 435 of the amino acid sequence ofSEQ ID NO:10, amino acid residues 474 to 677 of SEQ ID NO:2, or aminoacid residues 457 to 673 of SEQ ID NO:10. Nucleic acid moleculesencoding such peptides and polypeptides are useful as polymerase chainreaction primers and probes.

7. Production of Zcytor14 Fusion Proteins and Conjugates

One general class of Zcytor14 analogs are variants having an amino acidsequence that is a mutation of the amino acid sequence disclosed herein.Another general class of Zcytor14 analogs is provided by anti-idiotypeantibodies, and fragments thereof, as described below. Moreover,recombinant antibodies comprising anti-idiotype variable domains can beused as analogs (see, for example, Monfardini et al., Proc. Assoc. Am.Physicians 108:420 (1996)). Since the variable domains of anti-idiotypeZcytor14 antibodies mimic Zcytor14, these domains can provide Zcytor14binding activity. Methods of producing anti-idiotypic catalyticantibodies are known to those of skill in the art (see, for example,Joron et al., Ann. NY Acad. Sci. 672:216 (1992), Friboulet et al., Appl.Biochem. Biotechnol. 47:229 (1994), and Avalle et al., Ann. NY Acad.Sci. 864:118 (1998)).

Another approach to identifying Zcytor14 analogs is provided by the useof combinatorial libraries. Methods for constructing and screening phagedisplay and other combinatorial libraries are provided, for example, byKay et al., Phage Display of Peptides and Proteins (Academic Press1996), Verdine, U.S. Pat. No. 5,783,384, Kay, et. al., U.S. Pat. No.5,747,334, and Kauffman et al., U.S. Pat. No. 5,723,323.

Zcytor14 polypeptides have both in vivo and in vitro uses. As anillustration, a soluble form of Zcytor14 can be added to cell culturemedium to inhibit the effects of the Zcytor14 ligand produced by thecultured cells.

Fusion proteins of Zcytor14 can be used to express Zcytor14 in arecombinant host, and to isolate the produced Zcytor14. As describedbelow, particular Zcytor14 fusion proteins also have uses in diagnosisand therapy. One type of fusion protein comprises a peptide that guidesa Zcytor14 polypeptide from a recombinant host cell. To direct aZcytor14 polypeptide into the secretory pathway of a eukaryotic hostcell, a secretory signal sequence (also known as a signal peptide, aleader sequence, prepro sequence or pre sequence) is provided in theZcytor14 expression vector. While the secretory signal sequence may bederived from Zcytor14, a suitable signal sequence may also be derivedfrom another secreted protein or synthesized de novo. The secretorysignal sequence is operably linked to a Zcytor14-encoding sequence suchthat the two sequences are joined in the correct reading frame andpositioned to direct the newly synthesized polypeptide into thesecretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the nucleotide sequence encoding thepolypeptide of interest, although certain secretory signal sequences maybe positioned elsewhere in the nucleotide sequence of interest (see,e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat.No. 5,143,830).

Although the secretory signal sequence of Zcytor14 or another proteinproduced by mammalian cells (e.g., tissue-type plasminogen activatorsignal sequence, as described, for example, in U.S. Pat. No. 5,641,655)is useful for expression of Zcytor14 in recombinant mammalian hosts, ayeast signal sequence is preferred for expression in yeast cells.Examples of suitable yeast signal sequences are those derived from yeastmating phermone α-factor (encoded by the MFα1 gene), invertase (encodedby the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene). See,for example, Romanos et al., “Expression of Cloned Genes in Yeast,” inDNA Cloning 2: A Practical Approach, 2^(nd) Edition, Glover and Hames(eds.), pages 123-167 (Oxford University Press 1995).

In bacterial cells, it is often desirable to express a heterologousprotein as a fusion protein to decrease toxicity, increase stability,and to enhance recovery of the expressed protein. For example, Zcytor14can be expressed as a fusion protein comprising a glutathioneS-transferase polypeptide. Glutathione S-transferease fusion proteinsare typically soluble, and easily purifiable from E. coli lysates onimmobilized glutathione columns. In similar approaches, a Zcytor14fusion protein comprising a maltose binding protein polypeptide can beisolated with an amylose resin column, while a fusion protein comprisingthe C-terminal end of a truncated Protein A gene can be purified usingIgG-Sepharose. Established techniques for expressing a heterologouspolypeptide as a fusion protein in a bacterial cell are described, forexample, by Williams et al., “Expression of Foreign Proteins in E. coliUsing Plasmid Vectors and Purification of Specific PolyclonalAntibodies,” in DNA Cloning 2: A Practical Approach, 2^(nd) Edition,Glover and Hames (Eds.), pages 15-58 (Oxford University Press 1995). Inaddition, commercially available expression systems are available. Forexample, the PINPOINT Xa protein purification system (PromegaCorporation; Madison, Wis.) provides a method for isolating a fusionprotein comprising a polypeptide that becomes biotinylated duringexpression with a resin that comprises avidin.

Peptide tags that are useful for isolating heterologous polypeptidesexpressed by either prokaryotic or eukaryotic cells includepolyHistidine tags (which have an affinity for nickel-chelating resin),c-myc tags, calmodulin binding protein (isolated with calmodulinaffinity chromatography), substance P, the RYIRS tag (which binds withanti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which bindswith anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.Biophys. 329:215 (1996), Morganti et al., Biotechnol. Appl. Biochem.23:67 (1996), and Zheng et al., Gene 186:55 (1997). Nucleic acidmolecules encoding such peptide tags are available, for example, fromSigma-Aldrich Corporation (St. Louis, Mo.).

The present invention also contemplates that the use of the secretorysignal sequence contained in the Zcytor14 polypeptides of the presentinvention to direct other polypeptides into the secretory pathway. Asignal fusion polypeptide can be made wherein a secretory signalsequence derived from amino acid residues 1 to 20 of SEQ ID NO:2 isoperably linked to another polypeptide using methods known in the artand disclosed herein. The secretory signal sequence contained in thefusion polypeptides of the present invention is preferably fusedamino-terminally to an additional peptide to direct the additionalpeptide into the secretory pathway. Such constructs have numerousapplications known in the art. For example, these novel secretory signalsequence fusion constructs can direct the secretion of an activecomponent of a normally non-secreted protein, such as a receptor. Suchfusions may be used in a transgenic animal or in a cultured recombinanthost to direct peptides through the secretory pathway. With regard tothe latter, exemplary polypeptides include pharmaceutically activemolecules such as Factor VIIa, proinsulin, insulin, follicle stimulatinghormone, tissue type plasminogen activator, tumor necrosis factor,interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, and IL-18), colony stimulating factors (e.g., granulocyte-colonystimulating factor, and granulocyte macrophage-colony stimulatingfactor), interferons (e.g., interferons-α, -β, -γ, -ω, -δ, -□, and -τ),the stem cell growth factor designated “S1 factor,” erythropoietin, andthrombopoietin. The Zcytor14 secretory signal sequence contained in thefusion polypeptides of the present invention is preferably fusedamino-terminally to an additional peptide to direct the additionalpeptide into the secretory pathway. Fusion proteins comprising aZcytor14 secretory signal sequence can be constructed using standardtechniques.

Another form of fusion protein comprises a Zcytor14 polypeptide and animmunoglobulin heavy chain constant region, typically an F_(c) fragment,which contains two or three constant region domains and a hinge regionbut lacks the variable region. As an illustration, Chang et a., U.S.Pat. No. 5,723,125, describe a fusion protein comprising a humaninterferon and a human immunoglobulin Fc fragment. The C-terminal of theinterferon is linked to the N-terminal of the Fc fragment by a peptidelinker moiety. An example of a peptide linker is a peptide comprisingprimarily a T cell inert sequence, which is immunologically inert. Anexemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGGS (SEQ ID NO:7). In this fusion protein, a preferred Fc moiety is ahuman γ4 chain, which is stable in solution and has little or nocomplement activating activity. Accordingly, the present inventioncontemplates a Zcytor14 fusion protein that comprises a Zcytor14 moietyand a human Fc fragment, wherein the C-terminus of the Zcytor14 moietyis attached to the N-terminus of the Fc fragment via a peptide linker,such as a peptide consisting of the amino acid sequence of SEQ ID NO:7.The Zcytor14 moiety can be a Zcytor14 molecule or a fragment thereof.For example, a fusion protein can comprise a fragment of Zcytor14 thatcontains the extracellular domain (e.g., a soluble Zcytor14 receptor)and an Fc fragment (e.g., a human Fc fragment).

In another variation, a Zcytor14 fusion protein comprises an IgGsequence, a Zcytor14 moiety covalently joined to the aminoterminal endof the IgG sequence, and a signal peptide that is covalently joined tothe aminoterminal of the Zcytor14 moiety, wherein the IgG sequenceconsists of the following elements in the following order: a hingeregion, a CH₂ domain, and a CH₃ domain. Accordingly, the IgG sequencelacks a CH₁ domain. The Zcytor14 moiety displays a Zcytor14 activity, asdescribed herein, such as the ability to bind with a Zcytor14 ligand.This general approach to producing fusion proteins that comprise bothantibody and nonantibody portions has been described by LaRochelle etal., EP 742830 (WO 95/21258).

Fusion proteins comprising a Zcytor14 moiety and an Fc moiety can beused, for example, as an in vitro assay tool. For example, the presenceof a Zcytor14 ligand in a biological sample can be detected using aZcytor14-immunoglobulin fusion protein, in which the Zcytor14 moiety isused to bind the ligand, and a macromolecule, such as Protein A oranti-Fc antibody, is used to bind the fusion protein to a solid support.Such systems can be used to identify agonists and antagonists thatinterfere with the binding of a Zcytor14 ligand to its receptor.

Other examples of antibody fusion proteins include polypeptides thatcomprise an antigen-binding domain and a Zcytor14 fragment that containsa Zcytor14 extracellular domain. Such molecules can be used to targetparticular tissues for the benefit of Zcytor14 binding activity.

The present invention further provides a variety of other polypeptidefusions. For example, part or all of a domain(s) conferring a biologicalfunction can be swapped between Zcytor14 of the present invention withthe functionally equivalent domain(s) from another member of thecytokine receptor family. Polypeptide fusions can be expressed inrecombinant host cells to produce a variety of Zcytor14 fusion analogs.A Zcytor14 polypeptide can be fused to two or more moieties or domains,such as an affinity tag for purification and a targeting domain.Polypeptide fusions can also comprise one or more cleavage sites,particularly between domains. See, for example, Tuan et al., ConnectiveTissue Research 34:1 (1996).

Fusion proteins can be prepared by methods known to those skilled in theart by preparing each component of the fusion protein and chemicallyconjugating them. Alternatively, a polynucleotide encoding bothcomponents of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. General methods for enzymatic and chemical cleavage of fusionproteins are described, for example, by Ausubel (1995) at pages 16-19 to16-25.

Zcytor14 polypeptides can be used to identify and to isolate Zcytor14ligands. Zcytor14 extracellular domain (e.g., amino acid residues 1 to452, or 21 to 452 of SEQ ID NO:2) and other forms of a soluble Zcytor14receptor, are particularly useful for these methods. For example,proteins and peptides of the present invention can be immobilized on acolumn and used to bind ligands from a biological sample that is runover the column (Hermanson et al. (eds.), Immobilized Affinity LigandTechniques, pages 195-202 (Academic Press 1992)).

The activity of a Zcytor14 polypeptide can be observed by asilicon-based biosensor microphysiometer, which measures theextracellular acidification rate or proton excretion associated withreceptor binding and subsequent physiologic cellular responses. Anexemplary device is the CYTOSENSOR Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. A variety of cellular responses,such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and receptor activation, and the like,can be measured by this method (see, for example, McConnell et al.,Science 257:1906 (1992), Pitchford et al., Meth. Enzymol. 228:84 (1997),Arimilli et al., J. Immunol. Meth. 212:49 (1998), Van Liefde et al.,Eur. J. Pharmacol. 346:87 (1998)). The microphysiometer can be used forassaying eukaryotic, prokaryotic, adherent, or non-adherent cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including agonists, ligands, or antagonists of Zcytor14.

The microphysiometer can be used to measure responses of anZcytor14-expressing eukaryotic cell, compared to a control eukaryoticcell that does not express Zcytor14 polypeptide. Suitable cellsresponsive to Zcytor14-modulating stimuli include recombinant host cellscomprising a Zcytor14 expression vector, and cells that naturallyexpress Zcytor14. Extracellular acidification provides one measure for aZcytor14-modulated cellular response. In addition, this approach can beused to identify ligands, agonists, and antagonists of Zcytor14 ligand.For example, a molecule can be identified as an agonist of Zcytor14ligand by providing cells that express a Zcytor14 polypeptide, culturinga first portion of the cells in the absence of the test compound,culturing a second portion of the cells in the presence of the testcompound, and determining whether the second portion exhibits a cellularresponse, in comparison with the first portion.

Alternatively, a solid phase system can be used to identify a Zcytor14ligand, or an agonist or antagonist of a Zcytor14 ligand. For example, aZcytor14 polypeptide or Zcytor14 fusion protein can be immobilized ontothe surface of a receptor chip of a commercially available biosensorinstrument (BIACORE, Biacore AB; Uppsala, Sweden). The use of thisinstrument is disclosed, for example, by Karlsson, Immunol. Methods145:229 (1991), and Cunningham and Wells, J. Mol. Biol. 234:554 (1993).

In brief, a Zcytor14 polypeptide or fusion protein is covalentlyattached, using amine or sulfhydryl chemistry, to dextran fibers thatare attached to gold film within a flow cell. A test sample is thenpassed through the cell. If a ligand is present in the sample, it willbind to the immobilized polypeptide or fusion protein, causing a changein the refractive index of the medium, which is detected as a change insurface plasmon resonance of the gold film. This system allows thedetermination of on- and off-rates, from which binding affinity can becalculated, and assessment of stoichiometry of binding. This system canalso be used to examine antibody-antigen interactions, and theinteractions of other complement/anti-complement pairs.

Zcytor14 binding domains can be further characterized by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids of Zcytor14 ligand agonists. See, for example, de Voset al., Science 255:306 (1992), Smith et al., J. Mol. Biol. 224:899(1992), and Wlodaver et al., FEBS Lett. 309:59 (1992).

The present invention also contemplates chemically modified Zcytor14compositions, in which a Zcytor14 polypeptide is linked with a polymer.Illustrative Zcytor14 polypeptides are soluble polypeptides that lack afunctional transmembrane domain. Typically, the polymer is water solubleso that the Zcytor14 conjugate does not precipitate in an aqueousenvironment, such as a physiological environment. An example of asuitable polymer is one that has been modified to have a single reactivegroup, such as an active ester for acylation, or an aldehyde foralkylation. In this way, the degree of polymerization can be controlled.An example of a reactive aldehyde is polyethylene glycolpropionaldehyde, or mono-(C1-C10) alkoxy, or aryloxy derivatives thereof(see, for example, Harris, et al., U.S. Pat. No. 5,252,714). The polymermay be branched or unbranched. Moreover, a mixture of polymers can beused to produce Zcytor14 conjugates.

Zcytor14 conjugates used for therapy can comprise pharmaceuticallyacceptable water-soluble polymer moieties. Suitable water-solublepolymers include polyethylene glycol (PEG), monomethoxy-PEG,mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG,tresyl monomethoxy PEG, PEG propionaldehyde, bis-succinimidyl carbonatePEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxideco-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, dextran, cellulose, or other carbohydrate-based polymers.Suitable PEG may have a molecular weight from about 600 to about 60,000,including, for example, 5,000, 12,000, 20,000 and 25,000. A Zcytor14conjugate can also comprise a mixture of such water-soluble polymers.

One example of a Zcytor14 conjugate comprises a Zcytor14 moiety and apolyalkyl oxide moiety attached to the N-terminus of the Zcytor14moiety. PEG is one suitable polyalkyl oxide. As an illustration,Zcytor14 can be modified with PEG, a process known as “PEGylation.”PEGylation of Zcytor14 can be carried out by any of the PEGylationreactions known in the art (see, for example, EP 0 154 316, Delgado etal., Critical Reviews in Therapeutic Drug Carrier Systems 9:249 (1992),Duncan and Spreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis etal., Int J Hematol 68:1 (1998)). For example, PEGylation can beperformed by an acylation reaction or by an alkylation reaction with areactive polyethylene glycol molecule. In an alternative approach,Zcytor14 conjugates are formed by condensing activated PEG, in which aterminal hydroxy or amino group of PEG has been replaced by an activatedlinker (see, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with a Zcytor14 polypeptide. An example of anactivated PEG ester is PEG esterified to N-hydroxysuccinimide. As usedherein, the term “acylation” includes the following types of linkagesbetween Zcytor14 and a water soluble polymer: amide, carbamate,urethane, and the like. Methods for preparing PEGylated Zcytor14 byacylation will typically comprise the steps of (a) reacting a Zcytor14polypeptide with PEG (such as a reactive ester of an aldehyde derivativeof PEG) under conditions whereby one or more PEG groups attach toZcytor14, and (b) obtaining the reaction product(s). Generally, theoptimal reaction conditions for acylation reactions will be determinedbased upon known parameters and desired results. For example, the largerthe ratio of PEG:Zcytor14, the greater the percentage of polyPEGylatedZcytor14 product.

The product of PEGylation by acylation is typically a polyPEGylatedZcytor14 product, wherein the lysine ε-amino groups are PEGylated via anacyl linking group. An example of a connecting linkage is an amide.Typically, the resulting Zcytor14 will be at least 95% mono-, di-, ortri-pegylated, although some species with higher degrees of PEGylationmay be formed depending upon the reaction conditions. PEGylated speciescan be separated from unconjugated Zcytor14 polypeptides using standardpurification methods, such as dialysis, ultrafiltration, ion exchangechromatography, affinity chromatography, and the like.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with Zcytor14 in the presence of a reducing agent. PEGgroups are preferably attached to the polypeptide via a —CH₂—NH group.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the ε-amino groups of the lysine residuesand the α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups. The presentinvention provides a substantially homogenous preparation of Zcytor14monopolymer conjugates.

Reductive alkylation to produce a substantially homogenous population ofmonopolymer Zcytor14 conjugate molecule can comprise the steps of: (a)reacting a Zcytor14 polypeptide with a reactive PEG under reductivealkylation conditions at a pH suitable to permit selective modificationof the α-amino group at the amino terminus of the Zcytor14, and (b)obtaining the reaction product(s). The reducing agent used for reductivealkylation should be stable in aqueous solution and preferably be ableto reduce only the Schiff base formed in the initial process ofreductive alkylation. Preferred reducing agents include sodiumborohydride, sodium cyanoborohydride, dimethylamine borane,trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopolymer Zcytor14conjugates, the reductive alkylation reaction conditions are those whichpermit the selective attachment of the water soluble polymer moiety tothe N-terminus of Zcytor14. Such reaction conditions generally providefor pKa differences between the lysine amino groups and the α-aminogroup at the N-terminus. The pH also affects the ratio of polymer toprotein to be used. In general, if the pH is lower, a larger excess ofpolymer to protein will be desired because the less reactive theN-terminal α-group, the more polymer is needed to achieve optimalconditions. If the pH is higher, the polymer:Zcytor14 need not be aslarge because more reactive groups are available. Typically, the pH willfall within the range of 3-9,or 3-6.

Another factor to consider is the molecular weight of the water-solublepolymer. Generally, the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.For PEGylation reactions, the typical molecular weight is about 2 kDa toabout 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25kDa. The molar ratio of water-soluble polymer to Zcytor14 will generallybe in the range of 1:1 to 100:1. Typically, the molar ratio ofwater-soluble polymer to Zcytor14 will be 1:1 to 20:1 forpolyPEGylation, and 1:1 to 5:1 for monoPEGylation.

General methods for producing conjugates comprising a polypeptide andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al., U.S. Pat.No. 5,738,846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996),Monkarsh et al., Anal. Biochem. 247:434 (1997)).

The present invention contemplates compositions comprising a peptide orpolypeptide described herein. Such compositions can further comprise acarrier. The carrier can be a conventional organic or inorganic carrier.Examples of carriers include water, buffer solution, alcohol, propyleneglycol, macrogol, sesame oil, corn oil, and the like.

8. Isolation of Zcytor14 Polypeptides

The polypeptides of the present invention can be purified to at leastabout 80% purity, to at least about 90% purity, to at least about 95%purity, or even greater than 95% purity with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. The polypeptides of the presentinvention may also be purified to a pharmaceutically pure state, whichis greater than 99.9% pure. In certain preparations, a purifiedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin.

Fractionation and/or conventional purification methods can be used toobtain preparations of Zcytor14 purified from natural sources (e.g.,prostate or thyroid tissue), synthetic Zcytor14 polypeptides, andrecombinant Zcytor14 polypeptides and fusion Zcytor14 polypeptidespurified from recombinant host cells. In general, ammonium sulfateprecipitation and acid or chaotrope extraction may be used forfractionation of samples. Exemplary purification steps may includehydroxyapatite, size exclusion, FPLC and reverse-phase high performanceliquid chromatography. Suitable chromatographic media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.Exemplary chromatographic media include those media derivatized withphenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties.

Examples of coupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers. Selection of a particular method for polypeptide isolationand purification is a matter of routine design and is determined in partby the properties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988),and Doonan, Protein Purification Protocols (The Humana Press 1996).

Additional variations in Zcytor14 isolation and purification can bedevised by those of skill in the art. For example, anti-Zcytor14antibodies, obtained as described below, can be used to isolate largequantities of protein by immunoaffinity purification.

The polypeptides of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (M.Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification.

Zcytor14 polypeptides or fragments thereof may also be prepared throughchemical synthesis, as described above. Zcytor14 polypeptides may bemonomers or multimers; glycosylated or non-glycosylated; PEGylated ornon-PEGylated; and may or may not include an initial methionine aminoacid residue.

9. Production of Antibodies to Zcytor14 Proteins

Antibodies to Zcytor14 can be obtained, for example, using the productof a Zcytor14 expression vector or Zcytor14 isolated from a naturalsource as an antigen. Particularly useful anti-Zcytor14 antibodies “bindspecifically” with Zcytor14. Antibodies are considered to bespecifically binding if the antibodies exhibit at least one of thefollowing two properties: (1) antibodies bind to Zcytor14 with athreshold level of binding activity, and (2) antibodies do notsignificantly cross-react with polypeptides related to Zcytor14.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a Zcytor14 polypeptide, peptide or epitope with a bindingaffinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater,more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ orgreater. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, Ann. NY Acad. Sci. 51:660 (1949)). With regard to the secondcharacteristic, antibodies do not significantly cross-react with relatedpolypeptide molecules, for example, if they detect Zcytor14, but notpresently known polypeptides using a standard Western blot analysis.Examples of known related polypeptides include known cytokine receptors.

Anti-Zcytor14 antibodies can be produced using antigenic Zcytor14epitope-bearing peptides and polypeptides. Antigenic epitope-bearingpeptides and polypeptides of the present invention contain a sequence ofat least nine, or between 15 to about 30 amino acids contained withinSEQ ID NO:2 or another amino acid sequence disclosed herein. However,peptides or polypeptides comprising a larger portion of an amino acidsequence of the invention, containing from 30 to 50 amino acids, or anylength up to and including the entire amino acid sequence of apolypeptide of the invention, also are useful for inducing antibodiesthat bind with Zcytor14. It is desirable that the amino acid sequence ofthe epitope-bearing peptide is selected to provide substantialsolubility in aqueous solvents (i.e., the sequence includes relativelyhydrophilic residues, while hydrophobic residues are preferablyavoided). Moreover, amino acid sequences containing proline residues maybe also be desirable for antibody production.

As an illustration, potential antigenic sites in Zcytor14 wereidentified using the Jameson-Wolf method, Jameson and Wolf, CABIOS4:181, (1988), as implemented by the PROTEAN program (version 3.14) ofLASERGENE (DNASTAR; Madison, Wis.). Default parameters were used in thisanalysis.

The Jameson-Wolf method predicts potential antigenic determinants bycombining six major subroutines for protein structural prediction.Briefly, the Hopp-Woods method, Hopp et al., Proc. Nat'l Acad. Sci. USA78:3824 (1981), was first used to identify amino acid sequencesrepresenting areas of greatest local hydrophilicity (parameter: sevenresidues averaged). In the second step, Emini's method, Emini et al., J.Virology 55:836 (1985), was used to calculate surface probabilities(parameter: surface decision threshold (0.6)=1). Third, theKarplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212(1985), was used to predict backbone chain flexibility (parameter:flexibility threshold (0.2)=1). In the fourth and fifth steps of theanalysis, secondary structure predictions were applied to the data usingthe methods of Chou-Fasman, Chou, “Prediction of Protein StructuralClasses from Amino Acid Composition,” in Prediction of Protein Structureand the Principles of Protein Conformation, Fasman (ed.), pages 549-586(Plenum Press 1990), and Garnier-Robson, Garnier et al., J. Mol. Biol.120:97 (1978) (Chou-Fasman parameters: conformation table=64 proteins; αregion threshold=103; β region threshold=105; Garnier-Robson parameters:α and β decision constants=0). In the sixth subroutine, flexibilityparameters and hydropathy/solvent accessibility factors were combined todetermine a surface contour value, designated as the “antigenic index.”Finally, a peak broadening function was applied to the antigenic index,which broadens major surface peaks by adding 20, 40, 60, or 80% of therespective peak value to account for additional free energy derived fromthe mobility of surface regions relative to interior regions. Thiscalculation was not applied, however, to any major peak that resides ina helical region, since helical regions tend to be less flexible.

The results of this analysis indicated that the following amino acidsequences of SEQ ID NO:2 would provide suitable antigenic peptides:amino acids 26 to 33 (“antigenic peptide 1”), amino acids 41 to 46(“antigenic peptide 2”), 74 to 81 (“antigenic peptide 3”), amino acids95 to 105 (“antigenic peptide 4”), amino acids 109 to 119 (“antigenicpeptide 5”), amino acids 95 to 119 (“antigenic peptide 6”), amino acids178 to 185 (“antigenic peptide 7”), amino acids 200 to 206 (“antigenicpeptide 8”), amino acids 231 to 238 (“antigenic peptide 9”), amino acids231 to 241 (“antigenic peptide 10”), amino acids 264 to 270 (“antigenicpeptide 11”), amino acids 274 to 281 (“antigenic peptide 12”), aminoacids 317 to 324 (“antigenic peptide 13”), amino acids 357 to 363(“antigenic peptide 14”), amino acids 384 to 392 (“antigenic peptide15”), amino acids 398 to 411 (“antigenic peptide 16”), amino acids 405to 411 (“antigenic peptide 17”), amino acids 423 to 429 (“antigenicpeptide 18”), and amino acids 434 to 439 (“antigenic peptide 19”). Thepresent invention contemplates the use of any one of antigenic peptides1 to 19 to generate antibodies to Zcytor14. The present invention alsocontemplates polypeptides comprising at least one of antigenic peptides1 to 19.

Polyclonal antibodies to recombinant Zcytor14 protein or to Zcytor14isolated from natural sources can be prepared using methods well-knownto those of skill in the art. See, for example, Green et al.,“Production of Polyclonal Antisera,” in Immunochemical Protocols(Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995). The immunogenicity of a Zcytor14 polypeptide canbe increased through the use of an adjuvant, such as alum (aluminumhydroxide) or Freund's complete or incomplete adjuvant. Polypeptidesuseful for immunization also include fusion polypeptides, such asfusions of Zcytor14 or a portion thereof with an immunoglobulinpolypeptide or with maltose binding protein. The polypeptide immunogenmay be a full-length molecule or a portion thereof. If the polypeptideportion is “hapten-like,” such portion may be advantageously joined orlinked to a macromolecular carrier (such as keyhole limpet hemocyanin(KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such ashorses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, orsheep, an anti-Zcytor14 antibody of the present invention may also bederived from a subhuman primate antibody. General techniques for raisingdiagnostically and therapeutically useful antibodies in baboons may befound, for example, in Goldenberg et al., international patentpublication No. WO 91/11465, and in Losman et al., Int. J. Cancer 46:310(1990).

Alternatively, monoclonal anti-Zcytor14 antibodies can be generated.Rodent monoclonal antibodies to specific antigens may be obtained bymethods known to those skilled in the art (see, for example, Kohler etal., Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols inImmunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)[“Coligan”], Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a Zcytor14 gene product, verifying the presenceof antibody production by removing a serum sample, removing the spleento obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive clonesthat produce antibodies to the antigen, culturing the clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

In addition, an anti-Zcytor14 antibody of the present invention may bederived from a human monoclonal antibody. Human monoclonal antibodiesare obtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described, for example, by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994).

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-Zcytor14 antibodies. Such antibody fragments can be obtained, forexample, by proteolytic hydrolysis of the antibody. Antibody fragmentscan be obtained by pepsin or papain digestion of whole antibodies byconventional methods. As an illustration, antibody fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent to produce 3.5S Fab′ monovalent fragments.Optionally, the cleavage reaction can be performed using a blockinggroup for the sulfhydryl groups that result from cleavage of disulfidelinkages. As an alternative, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem.J. 73:119 (1959), Edelman et al., in Methods in Enzymology Vol. 1, page422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992)).

The Fv fragments may comprise V_(H) and V_(L) chains, which areconnected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains which areconnected by an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cell,such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are described, for example, by Whitlow etal., Methods: A Companion to Methods in Enzymology 2:97 (1991) (alsosee, Bird et al., Science 242:423 (1988), Ladner et al., U.S. Pat. No.4,946,778, Pack et al., Bio/Technology 11:1271 (1993), and Sandhu,supra).

As an illustration, a scFV can be obtained by exposing lymphocytes toZcytor14 polypeptide in vitro, and selecting antibody display librariesin phage or similar vectors (for instance, through use of immobilized orlabeled Zcytor14 protein or peptide). Genes encoding polypeptides havingpotential Zcytor14 polypeptide binding domains can be obtained byscreening random peptide libraries displayed on phage (phage display) oron bacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. These random peptidedisplay libraries can be used to screen for peptides, which interactwith a known target which can be a protein or polypeptide, such as aligand or receptor, a biological or synthetic macromolecule, or organicor inorganic substances. Techniques for creating and screening suchrandom peptide display libraries are known in the art (Ladner et al.,U.S. Pat. No. 5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladneret al., U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698,and Kay et al., Phage Display of Peptides and Proteins (Academic Press,Inc. 1996)) and random peptide display libraries and kits for screeningsuch libraries are available commercially, for instance from CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego,Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKBBiotechnology Inc. (Piscataway, N.J.). Random peptide display librariescan be screened using the Zcytor14 sequences disclosed herein toidentify proteins which bind to Zcytor14.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-Zcytor14 antibody may be derived from a“humanized” monoclonal antibody. Humanized monoclonal antibodies areproduced by transferring mouse complementary determining regions fromheavy and light variable chains of the mouse immunoglobulin into a humanvariable domain. Typical residues of human antibodies are thensubstituted in the framework regions of the murine counterparts. The useof antibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Nat'l Acad. Sci. USA 86:3833 (1989). Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522 (1986), Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285(1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer et al., J.Immun. 150:2844 (1993), Sudhir (ed.), Antibody Engineering Protocols(Humana Press, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,”in Protein Engineering: Principles and Practice, Cleland et al. (eds.),pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S.Pat. No. 5,693,762 (1997).

Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-Zcytor14 antibodies or antibody fragments, usingstandard techniques. See, for example, Green et al., “Production ofPolyclonal Antisera,” in Methods In Molecular Biology: ImmunochemicalProtocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, seeColigan at pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotypeantibodies can be prepared using anti-Zcytor14 antibodies or antibodyfragments as immunogens with the techniques, described above. As anotheralternative, humanized anti-idiotype antibodies or subhuman primateanti-idiotype antibodies can be prepared using the above-describedtechniques. Methods for producing anti-idiotype antibodies aredescribed, for example, by Irie, U.S. Pat. No. 5,208,146, Greene, et.al., U.S. Pat. No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol.77:1875 (1996).

10. Use of Zcytor14 Nucleotide Sequences to Detect Gene Expression andGene Structure

Nucleic acid molecules can be used to detect the expression of aZcytor14 gene in a biological sample. Certain probe molecules includedouble-stranded nucleic acid molecules comprising the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:4, or a portion thereof, as well assingle-stranded nucleic acid molecules having the complement of thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, or a portion thereof.Probe molecules may be DNA, RNA, oligonucleotides, and the like. As usedherein, the term “portion” refers to at least eight nucleotides to atleast 20 or more nucleotides. Certain probes bind with regions of theZcytor14 gene that have a low sequence similarity to comparable regionsin other cytokine receptor genes.

In a basic assay, a single-stranded probe molecule is incubated withRNA, isolated from a biological sample, under conditions of temperatureand ionic strength that promote base pairing between the probe andtarget Zcytor14 RNA species. After separating unbound probe fromhybridized molecules, the amount of hybrids is detected.

Well-established hybridization methods of RNA detection include northernanalysis and dot/slot blot hybridization (see, for example, Ausubel(1995) at pages 4-1 to 4-27, and Wu et al. (eds.), “Analysis of GeneExpression at the RNA Level,” in Methods in Gene Biotechnology, pages225-239 (CRC Press, Inc. 1997)). Nucleic acid probes can be detectablylabeled with radioisotopes such as ³²P or ³⁵S. Alternatively, Zcytor14RNA can be detected with a nonradioactive hybridization method (see, forexample, Isaac (ed.), Protocols for Nucleic Acid Analysis byNonradioactive Probes (Humana Press, Inc. 1993)). Typically,nonradioactive detection is achieved by enzymatic conversion ofchromogenic or chemiluminescent substrates. Illustrative nonradioactivemoieties include biotin, fluorescein, and digoxigenin.

Zcytor14 oligonucleotide probes are also useful for in vivo diagnosis.As an illustration, ¹⁸F-labeled oligonucleotides can be administered toa subject and visualized by positron emission tomography (Tavitian etal., Nature Medicine 4:467 (1998)).

Numerous diagnostic procedures take advantage of the polymerase chainreaction (PCR) to increase sensitivity of detection methods. Standardtechniques for performing PCR are well-known (see, generally, Mathew(ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),White (ed.), PCR Protocols: Current Methods and Applications (HumanaPress, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (HumanaPress, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (HumanaPress, Inc. 1998)).

PCR primers can be designed to amplify a portion of the Zcytor14 genethat has a low sequence similarity to a comparable region in otherproteins, such as other cytokine receptor proteins.

One variation of PCR for diagnostic assays is reverse transcriptase-PCR(RT-PCR). In the RT-PCR technique, RNA is isolated from a biologicalsample, reverse transcribed to cDNA, and the cDNA is incubated withZcytor14 primers (see, for example, Wu et al. (eds.), “Rapid Isolationof Specific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology,pages 15-28 (CRC Press, Inc. 1997)). PCR is then performed and theproducts are analyzed using standard techniques.

As an illustration, RNA is isolated from biological sample using, forexample, the guanidinium-thiocyanate cell lysis procedure describedabove. Alternatively, a solid-phase technique can be used to isolatemRNA from a cell lysate. A reverse transcription reaction can be primedwith the isolated RNA using random oligonucleotides, short homopolymersof dT, or Zcytor14 anti-sense oligomers. Oligo-dT primers offer theadvantage that various mRNA nucleotide sequences are amplified that canprovide control target sequences. Zcytor14 sequences are amplified bythe polymerase chain reaction using two flanking oligonucleotide primersthat are typically 20 bases in length.

PCR amplification products can be detected using a variety ofapproaches. For example, PCR products can be fractionated by gelelectrophoresis, and visualized by ethidium bromide staining.Alternatively, fractionated PCR products can be transferred to amembrane, hybridized with a detectably-labeled Zcytor14 probe, andexamined by autoradiography. Additional alternative approaches includethe use of digoxigenin-labeled deoxyribonucleic acid triphosphates toprovide chemiluminescence detection, and the C-TRAK colorimetric assay.

Another approach for detection of Zcytor14 expression is cycling probetechnology (CPT), in which a single-stranded DNA target binds with anexcess of DNA-RNA-DNA chimeric probe to form a complex, the RNA portionis cleaved with RNAase H, and the presence of cleaved chimeric probe isdetected (see, for example, Beggs et al., J. Clin. Microbiol. 34:2985(1996), Bekkaoui et al., Biotechniques 20:240 (1996)). Alternativemethods for detection of Zcytor14 sequences can utilize approaches suchas nucleic acid sequence-based amplification, cooperative amplificationof templates by cross-hybridization, and the ligase chain reaction (see,for example, Marshall et al., U.S. Pat. No. 5,686,272 (1997), Dyer etal., J. Virol. Methods 60:161 (1996), Ehricht et al., Eur. J. Biochem.243:358 (1997), and Chadwick et al., J. Virol. Methods 70:59 (1998)).Other standard methods are known to those of skill in the art.

Zcytor14 probes and primers can also be used to detect and to localizeZcytor14 gene expression in tissue samples. Methods for such in situhybridization are well-known to those of skill in the art (see, forexample, Choo (ed.), In Situ Hybridization Protocols (Humana Press, Inc.1994), Wu et al. (eds.), “Analysis of Cellular DNA or Abundance of mRNAby Radioactive In Situ Hybridization (RISH),” in Methods in GeneBiotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu et al.(eds.), “Localization of DNA or Abundance of mRNA by Fluorescence InSitu Hybridization (RISH),” in Methods in Gene Biotechnology, pages279-289 (CRC Press, Inc. 1997)). Various additional diagnosticapproaches are well-known to those of skill in the art (see, forexample, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), Coleman and Tsongalis, Molecular Diagnostics (HumanaPress, Inc. 1996), and Elles, Molecular Diagnosis of Genetic Diseases(Humana Press, Inc., 1996)). Suitable test samples include blood, urine,saliva, tissue biopsy, and autopsy material.

The Zcytor14 gene resides in human chromosome 3p25-3p24. This region isassociated with various disorders, including xeroderma pigmentosum,Marfan-like connective tissue disorder, cardiomyopathy, diabetesmellitus, Fanconi anemia, renal cell carcinoma, Marfan syndrome, VonHippel-Lindau syndrome, and blepharophimosis. In addition, mutations ofcytokine receptors are associated with particular diseases. For example,polymorphisms of cytokine receptors are associated with pulmonaryalveolar proteinosis, familial periodic fever, and erythroleukemia.Thus, Zcytor14 nucleotide sequences can be used in linkage-based testingfor various diseases, and to determine whether a subject's chromosomescontain a mutation in the Zcytor14 gene. Detectable chromosomalaberrations at the Zcytor14 gene locus include, but are not limited to,aneuploidy, gene copy number changes, insertions, deletions, restrictionsite changes and rearrangements. Of particular interest are geneticalterations that inactivate a Zcytor14 gene.

Aberrations associated with the Zcytor14 locus can be detected usingnucleic acid molecules of the present invention by employing moleculargenetic techniques, such as restriction fragment length polymorphismanalysis, short tandem repeat analysis employing PCR techniques,amplification-refractory mutation system analysis, single-strandconformation polymorphism detection, RNase cleavage methods, denaturinggradient gel electrophoresis, fluorescence-assisted mismatch analysis,and other genetic analysis techniques known in the art (see, forexample, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis,Molecular Diagnostics (Human Press, Inc. 1996), Elles (ed.) MolecularDiagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren(ed.), Laboratory Protocols for Mutation Detection (Oxford UniversityPress 1996), Birren et al. (eds.), Genome Analysis, Vol. 2: DetectingGenes (Cold Spring Harbor Laboratory Press 1998), Dracopoli et al.(eds.), Current Protocols in Human Genetics (John Wiley & Sons 1998),and Richards and Ward, “Molecular Diagnostic Testing,” in Principles ofMolecular Medicine, pages 83-88 (Humana Press, Inc. 1998)).

The protein truncation test is also useful for detecting theinactivation of a gene in which translation-terminating mutationsproduce only portions of the encoded protein (see, for example,Stoppa-Lyonnet et al., Blood 91:3920 (1998)). According to thisapproach, RNA is isolated from a biological sample, and used tosynthesize cDNA. PCR is then used to amplify the Zcytor14 targetsequence and to introduce an RNA polymerase promoter, a translationinitiation sequence, and an in-frame ATG triplet. PCR products aretranscribed using an RNA polymerase, and the transcripts are translatedin vitro with a T7-coupled reticulocyte lysate system. The translationproducts are then fractionated by SDS-PAGE to determine the lengths ofthe translation products. The protein truncation test is described, forexample, by Dracopoli et al. (eds.), Current Protocols in HumanGenetics, pages 9.11.1-9.11.18 (John Wiley & Sons 1998).

The present invention also contemplates kits for performing a diagnosticassay for Zcytor14 gene expression or to detect mutations in theZcytor14 gene. Such kits comprise nucleic acid probes, such asdouble-stranded nucleic acid molecules comprising the nucleotidesequence of SEQ ID NO:1 or SEQ ID NO:4, or a portion thereof, as well assingle-stranded nucleic acid molecules having the complement of thenucleotide sequence of SEQ ID NO:1 or SEQ ID NO:4, or a portion thereof.Probe molecules may be DNA, RNA, oligonucleotides, and the like. Kitsmay comprise nucleic acid primers for performing PCR.

Such a kit can contain all the necessary elements to perform a nucleicacid diagnostic assay described above. A kit will comprise at least onecontainer comprising a Zcytor14 probe or primer. The kit may alsocomprise a second container comprising one or more reagents capable ofindicating the presence of Zcytor14 sequences. Examples of suchindicator reagents include detectable labels such as radioactive labels,fluorochromes, chemiluminescent agents, and the like. A kit may alsocomprise a means for conveying to the user that the Zcytor14 probes andprimers are used to detect Zcytor14 gene expression. For example,written instructions may state that the enclosed nucleic acid moleculescan be used to detect either a nucleic acid molecule that encodesZcytor14, or a nucleic acid molecule having a nucleotide sequence thatis complementary to a Zcytor14-encoding nucleotide sequence. The writtenmaterial can be applied directly to a container, or the written materialcan be provided in the form of a packaging insert.

11. Use of Anti-Zcytor14 Antibodies to Detect Zcytor14

The present invention contemplates the use of anti-Zcytor14 antibodiesto screen biological samples in vitro for the presence of Zcytor14. Inone type of in vitro assay, anti-Zcytor14 antibodies are used in liquidphase. For example, the presence of Zcytor14 in a biological sample canbe tested by mixing the biological sample with a trace amount of labeledZcytor14 and an anti-Zcytor14 antibody under conditions that promotebinding between Zcytor14 and its antibody. Complexes of Zcytor14 andanti-Zcytor14 in the sample can be separated from the reaction mixtureby contacting the complex with an immobilized protein which binds withthe antibody, such as an Fc antibody or Staphylococcus protein A. Theconcentration of Zcytor14 in the biological sample will be inverselyproportional to the amount of labeled Zcytor14 bound to the antibody anddirectly related to the amount of free labeled Zcytor14. Illustrativebiological samples include blood, urine, saliva, tissue biopsy, andautopsy material.

Alternatively, in vitro assays can be performed in which anti-Zcytor14antibody is bound to a solid-phase carrier. For example, antibody can beattached to a polymer, such as aminodextran, in order to link theantibody to an insoluble support such as a polymer-coated bead, a plateor a tube. Other suitable in vitro assays will be readily apparent tothose of skill in the art.

In another approach, anti-Zcytor14 antibodies can be used to detectZcytor14 in tissue sections prepared from a biopsy specimen. Suchimmunochemical detection can be used to determine the relative abundanceof Zcytor14 and to determine the distribution of Zcytor14 in theexamined tissue. General immunochemistry techniques are well established(see, for example, Ponder, “Cell Marking Techniques and TheirApplication,” in Mammalian Development: A Practical Approach, Monk(ed.), pages 115-38 (IRL Press 1987), Coligan at pages 5.8.1-5.8.8,Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley Interscience 1990), andManson (ed.), Methods In Molecular Biology, Vol. 10: ImmunochemicalProtocols (The Humana Press, Inc. 1992)).

Immunochemical detection can be performed by contacting a biologicalsample with an anti-Zcytor14 antibody, and then contacting thebiological sample with a detectably labeled molecule which binds to theantibody. For example, the detectably labeled molecule can comprise anantibody moiety that binds to anti-Zcytor14 antibody. Alternatively, theanti-Zcytor14 antibody can be conjugated with avidin/streptavidin (orbiotin) and the detectably labeled molecule can comprise biotin (oravidin/streptavidin). Numerous variations of this basic technique arewell-known to those of skill in the art.

Alternatively, an anti-Zcytor14 antibody can be conjugated with adetectable label to form an anti-Zcytor14 immunoconjugate. Suitabledetectable labels include, for example, a radioisotope, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescent labelor colloidal gold. Methods of making and detecting suchdetectably-labeled immunoconjugates are well-known to those of ordinaryskill in the art, and are described in more detail below.

The detectable label can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Anti-Zcytor14 immunoconjugates can also be labeled with a fluorescentcompound. The presence of a fluorescently-labeled antibody is determinedby exposing the immunoconjugate to light of the proper wavelength anddetecting the resultant fluorescence. Fluorescent labeling compoundsinclude fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Alternatively, anti-Zcytor14 immunoconjugates can be detectably labeledby coupling an antibody component to a chemiluminescent compound. Thepresence of the chemiluminescent-tagged immunoconjugate is determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of chemiluminescent labeling compoundsinclude luminol, isoluminol, an aromatic acridinium ester, an imidazole,an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label anti-Zcytor14immunoconjugates of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Bioluminescent compounds that are useful forlabeling include luciferin, luciferase and aequorin.

Alternatively, anti-Zcytor14 immunoconjugates can be detectably labeledby linking an anti-Zcytor14 antibody component to an enzyme. When theanti-Zcytor14-enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety, which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label polyspecific immunoconjugatesinclude β-galactosidase, glucose oxidase, peroxidase and alkalinephosphatase.

Those of skill in the art will know of other suitable labels, which canbe employed in accordance with the present invention. The binding ofmarker moieties to anti-Zcytor14 antibodies can be accomplished usingstandard techniques known to the art. Typical methodology in this regardis described by Kennedy et al., Clin. Chim. Acta 70:1 (1976), Schurs etal., Clin. Chim. Acta 81:1 (1977), Shih et al., Int'l J. Cancer 46:1101(1990), Stein et al, Cancer Res. 50:1330 (1990), and Coligan, supra.

Moreover, the convenience and versatility of immunochemical detectioncan be enhanced by using anti-Zcytor14 antibodies that have beenconjugated with avidin, streptavidin, and biotin (see, for example,Wilchek et al. (eds.), “Avidin-Biotin Technology,” Methods InEnzymology, Vol. 184 (Academic Press 1990), and Bayer et al.,“Immunochemical Applications of Avidin-Biotin Technology,” in Methods InMolecular Biology, Vol. 10, Manson (ed.), pages 149-162 (The HumanaPress, Inc. 1992).

Methods for performing immunoassays are well-established. See, forexample, Cook and Self, “Monoclonal Antibodies in DiagnosticImmunoassays,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 180-208,(Cambridge University Press, 1995), Perry, “The Role of MonoclonalAntibodies in the Advancement of Immunoassay Technology,” in MonoclonalAntibodies: Principles and Applications, Birch and Lennox (eds.), pages107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (AcademicPress, Inc. 1996).

The present invention also contemplates kits for performing animmunological diagnostic assay for Zcytor14 gene expression. Such kitscomprise at least one container comprising an anti-Zcytor14 antibody, orantibody fragment. A kit may also comprise a second container comprisingone or more reagents capable of indicating the presence of Zcytor14antibody or antibody fragments. Examples of such indicator reagentsinclude detectable labels such as a radioactive label, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescentlabel, colloidal gold, and the like. A kit may also comprise a means forconveying to the user that Zcytor14 antibodies or antibody fragments areused to detect Zcytor14 protein. For example, written instructions maystate that the enclosed antibody or antibody fragment can be used todetect Zcytor14. The written material can be applied directly to acontainer, or the written material can be provided in the form of apackaging insert.

12. Therapeutic Uses of Polypeptides having Zcytor14 Activity

The present invention includes the use of proteins, polypeptides, andpeptides having Zcytor14 activity (such as Zcytor14 polypeptides (e.g.,soluble forms of Zcytor14), Zcytor14 analogs (e.g., anti-Zcytor14anti-idiotype antibodies), and Zcytor14 fusion proteins) to a subjectwho lacks an adequate amount of this polypeptide. In contrast, Zcytor14antagonists (e.g., anti-Zcytor14 antibodies) can be used to treat asubject who produces an excess of Zcytor14.

As an illustration, Zcytor14 has an amino acid sequence that sharessimilarity with the human interleukin-17 receptor. Studies indicate thatinterleukin-17 plays a pivotal role in initiating or sustaining aninflammatory response (see, for example, Jovanovic et al., J. Immunol.160:3513 (1998)). Moreover, there is evidence that interleukin-17activates the production of inflammatory mediators by synoviocytes, andthat interleukin-17 contributes to the proinflammatory pattern that ischaracteristic of rheumatoid arthritis (Chabaud et al., J. Immunol.161:409 (1998); Chabaud et al., Arthritis Rheum. 42:963 (1999)).Accordingly, polypeptides having Zcytor14 activity (e.g., Zcytor14polypeptides, functional fragments of Zcytor14 including a solubleZcytor14 receptor, anti-Zcytor14 anti-idiotype antibodies, etc.) can beused to treat inflammation, and conditions, such as rheumatoidarthritis, that are associated with inflammation.

Generally, the dosage of administered Zcytor14 (or Zcytor14 analog orfusion protein) will vary depending upon such factors as the patient'sage, weight, height, sex, general medical condition and previous medicalhistory. Typically, it is desirable to provide the recipient with adosage of Zcytor14 polypeptide, which is in the range of from about 1pg/kg to 10 mg/kg (amount of agent/body weight of patient), although alower or higher dosage also may be administered as circumstancesdictate.

Administration of a Zcytor14 polypeptide to a subject can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringtherapeutic proteins by injection, the administration may be bycontinuous infusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprising Zcytor14can be prepared and inhaled with the aid of dry-powder dispersers,liquid aerosol generators, or nebulizers (e.g., Pettit and Gombotz,TIBTECH 16:343 (1998); Patton et al., Adv. Drug Deliv. Rev. 35:235(1999)). This approach is illustrated by the AERX diabetes managementsystem, which is a hand-held electronic inhaler that deliversaerosolized insulin into the lungs. Studies have shown that proteins aslarge as 48,000 kDa have been delivered across skin at therapeuticconcentrations with the aid of low-frequency ultrasound, whichillustrates the feasibility of trascutaneous administration (Mitragotriet al., Science 269:850 (1995)). Transdermal delivery usingelectroporation provides another means to administer a molecule havingZcytor14 binding activity (Potts et al., Pharm. Biotechnol. 10:213(1997)).

A pharmaceutical composition comprising a protein, polypeptide, orpeptide having Zcytor14 binding activity can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe therapeutic proteins are combined in a mixture with apharmaceutically acceptable carrier. A composition is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example, Gennaro(ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, molecules having Zcytor14 binding activity anda pharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a protein,polypeptide, or peptide having Zcytor14 binding activity and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. For example, an agent used to treat inflammation isphysiologically significant if its presence alleviates the inflammatoryresponse.

A pharmaceutical composition comprising Zcytor14 (or Zcytor14 analog orfusion protein) can be furnished in liquid form, in an aerosol, or insolid form. Liquid forms, are illustrated by injectable solutions andoral suspensions. Exemplary solid forms include capsules, tablets, andcontrolled-release forms. The latter form is illustrated by miniosmoticpumps and implants (Bremer et al., Pharm. Biotechnol. 10:239 (1997);Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems, Ranadeand Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al.,“Protein Delivery with Infusion Pumps,” in Protein Delivery: PhysicalSystems, Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997);Yewey et al., “Delivery of Proteins from a Controlled Release InjectableImplant,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 93-117 (Plenum Press 1997)).

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull.20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull.20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

Polypeptides having Zcytor14 binding activity can be encapsulated withinliposomes using standard techniques of protein microencapsulation (see,for example, Anderson et al., Infect. Immun. 31:1099 (1981), Anderson etal., Cancer Res. 50:1853 (1990), and Cohen et al., Biochim. Biophys.Acta 1063:95 (1991), Alving et al. “Preparation and Use of Liposomes inImmunological Studies,” in Liposome Technology, 2nd Edition, Vol. III,Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al., Meth.Enzymol. 149:124 (1987)). As noted above, therapeutically usefulliposomes may contain a variety of components. For example, liposomesmay comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,Biochim. Biophys. Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

The present invention also contemplates chemically modified polypeptideshaving binding Zcytor14 activity and Zcytor14 antagonists, in which apolypeptide is linked with a polymer, as discussed above.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises a polypeptide with a Zcytor14extracellular domain or a Zcytor14 antagonist (e.g., an antibody orantibody fragment that binds a Zcytor14 polypeptide). Therapeuticpolypeptides can be provided in the form of an injectable solution forsingle or multiple doses, or as a sterile powder that will bereconstituted before injection. Alternatively, such a kit can include adry-powder disperser, liquid aerosol generator, or nebulizer foradministration of a therapeutic polypeptide. Such a kit may furthercomprise written information on indications and usage of thepharmaceutical composition. Moreover, such information may include astatement that the Zcytor14 composition is contraindicated in patientswith known hypersensitivity to Zcytor14.

13. Therapeutic Uses of Zcytor14 Nucleotide Sequences

The present invention includes the use of Zcytor14 nucleotide sequencesto provide Zcytor14 to a subject in need of such treatment. In addition,a therapeutic expression vector can be provided that inhibits Zcytor14gene expression, such as an anti-sense molecule, a ribozyme, or anexternal guide sequence molecule.

There are numerous approaches to introduce a Zcytor14 gene to a subject,including the use of recombinant host cells that express Zcytor14,delivery of naked nucleic acid encoding Zcytor14, use of a cationiclipid carrier with a nucleic acid molecule that encodes Zcytor14, andthe use of viruses that express Zcytor14, such as recombinantretroviruses, recombinant adeno-associated viruses, recombinantadenoviruses, and recombinant Herpes simplex viruses (see, for example,Mulligan, Science 260:926 (1993), Rosenberg et al., Science 242:1575(1988), LaSalle et al., Science 259:988 (1993), Wolff et al., Science247:1465 (1990), Breakfield and Deluca, The New Biologist 3:203 (1991)).In an ex vivo approach, for example, cells are isolated from a subject,transfected with a vector that expresses a Zcytor14 gene, and thentransplanted into the subject.

In order to effect expression of a Zcytor14 gene, an expression vectoris constructed in which a nucleotide sequence encoding a Zcytor14 geneis operably linked to a core promoter, and optionally a regulatoryelement, to control gene transcription. The general requirements of anexpression vector are described above.

Alternatively, a Zcytor14 gene can be delivered using recombinant viralvectors, including for example, adenoviral vectors (e.g., Kass-Eisler etal., Proc. Nat'l Acad. Sci. USA 90:11498 (1993), Kolls et al., Proc.Nat'l Acad. Sci. USA 91:215 (1994), Li et al., Hum. Gene Ther. 4:403(1993), Vincent et al., Nat. Genet. 5:130 (1993), and Zabner et al.,Cell 75:207 (1993)), adenovirus-associated viral vectors (Flotte et al.,Proc. Nat'l Acad. Sci. USA 90:10613 (1993)), alphaviruses such asSemliki Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857(1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al.,Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Pat. Nos.4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus vectors(Koering et al., Hum. Gene Therap. 5:457 (1994)), pox virus vectors(Ozaki et al., Biochem. Biophys. Res. Comm. 193:653 (1993), Panicali andPaoletti, Proc. Nat'l Acad. Sci. USA 79:4927 (1982)), pox viruses, suchas canary pox virus or vaccinia virus (Fisher-Hoch et al., Proc. Nat'lAcad. Sci. USA 86:317 (1989), and Flexner et al., Ann. N.Y. Acad. Sci.569:86 (1989)), and retroviruses (e.g., Baba et al., J. Neurosurg 79:729(1993), Ram et al., Cancer Res. 53:83 (1993), Takamiya et al., J.Neurosci. Res 33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993),Vile and Hart, Cancer Res. 53:3860 (1993), and Anderson et al., U.S.Pat. No. 5,399,346). Within various embodiments, either the viral vectoritself, or a viral particle, which contains the viral vector may beutilized in the methods and compositions described below.

As an illustration of one system, adenovirus, a double-stranded DNAvirus, is a well-characterized gene transfer vector for delivery of aheterologous nucleic acid molecule (for a review, see Becker et al.,Meth. Cell Biol. 43:161 (1994); Douglas and Curiel, Science & Medicine4:44 (1997)). The adenovirus system offers several advantages including:(i) the ability to accommodate relatively large DNA inserts, (ii) theability to be grown to high-titer, (iii) the ability to infect a broadrange of mammalian cell types, and (iv) the ability to be used with manydifferent promoters including ubiquitous, tissue specific, andregulatable promoters. In addition, adenoviruses can be administered byintravenous injection, because the viruses are stable in thebloodstream.

Using adenovirus vectors where portions of the adenovirus genome aredeleted, inserts are incorporated into the viral DNA by direct ligationor by homologous recombination with a co-transfected plasmid. In anexemplary system, the essential E1 gene is deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell. When intravenously administered to intact animals,adenovirus primarily targets the liver. Although an adenoviral deliverysystem with an E1 gene deletion cannot replicate in the host cells, thehost's tissue will express and process an encoded heterologous protein.Host cells will also secrete the heterologous protein if thecorresponding gene includes a secretory signal sequence. Secretedproteins will enter the circulation from tissue that expresses theheterologous gene (e.g., the highly vascularized liver).

Moreover, adenoviral vectors containing various deletions of viral genescan be used to reduce or eliminate immune responses to the vector. Suchadenoviruses are E1-deleted, and in addition, contain deletions of E2Aor E4 (Lusky et al., J. Virol. 72:2022 (1998); Raper et al., Human GeneTherapy 9:671 (1998)). The deletion of E2b has also been reported toreduce immune responses (Amalfitano et al., J. Virol. 72:926 (1998)). Bydeleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. Generation of so called “gutless”adenoviruses, where all viral genes are deleted, are particularlyadvantageous for insertion of large inserts of heterologous DNA (for areview, see Yeh. and Perricaudet, FASEB J. 11:615 (1997)).

High titer stocks of recombinant viruses capable of expressing atherapeutic gene can be obtained from infected mammalian cells usingstandard methods. For example, recombinant herpes simplex virus can beprepared in Vero cells, as described by Brandt et al., J. Gen. Virol.72:2043 (1991), Herold et al., J. Gen. Virol. 75:1211 (1994), Visalliand Brandt, Virology 185:419 (1991), Grau et al., Invest. Ophthalmol.Vis. Sci. 30:2474 (1989), Brandt et al., J. Virol. Meth. 36:209 (1992),and by Brown and MacLean (eds.), HSV Virus Protocols (Humana Press1997).

Alternatively, an expression vector comprising a Zcytor14 gene can beintroduced into a subject's cells by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987); Mackey et al., Proc. Nat'lAcad. Sci. USA 85:8027 (1988)). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Liposomes can be used to direct transfection to particularcell types, which is particularly advantageous in a tissue with cellularheterogeneity, such as the pancreas, liver, kidney, and brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting. Targeted peptides (e.g., hormones or neurotransmitters),proteins such as antibodies, or non-peptide molecules can be coupled toliposomes chemically.

Electroporation is another alternative mode of administration. Forexample, Aihara and Miyazaki, Nature Biotechnology 16:867 (1998), havedemonstrated the use of in vivo electroporation for gene transfer intomuscle.

In an alternative approach to gene therapy, a therapeutic gene mayencode a Zcytor14 anti-sense RNA that inhibits the expression ofZcytor14. Suitable sequences for anti-sense molecules can be derivedfrom the nucleotide sequences of Zcytor14 disclosed herein.

Alternatively, an expression vector can be constructed in which aregulatory element is operably linked to a nucleotide sequence thatencodes a ribozyme. Ribozymes can be designed to express endonucleaseactivity that is directed to a certain target sequence in a mRNAmolecule (see, for example, Draper and Macejak, U.S. Pat. No. 5,496,698,McSwiggen, U.S. Pat. No. 5,525,468, Chowrira and McSwiggen, U.S. Pat.No. 5,631,359, and Robertson and Goldberg, U.S. Pat. No. 5,225,337). Inthe context of the present invention, ribozymes include nucleotidesequences that bind with Zcytor14 mRNA.

In another approach, expression vectors can be constructed in which aregulatory element directs the production of RNA transcripts capable ofpromoting RNase P-mediated cleavage of mRNA molecules that encode aZcytor14 gene. According to this approach, an external guide sequencecan be constructed for directing the endogenous ribozyme, RNase P, to aparticular species of intracellular mRNA, which is subsequently cleavedby the cellular ribozyme (see, for example, Altman et al., U.S. Pat. No.5,168,053, Yuan et al., Science 263:1269 (1994), Pace et al.,international publication No. WO 96/18733, George et al., internationalpublication No. WO 96/21731, and Werner et al., internationalpublication No. WO 97/33991). Preferably, the external guide sequencecomprises a ten to fifteen nucleotide sequence complementary to Zcytor14mRNA, and a 3′-NCCA nucleotide sequence, wherein N is preferably apurine. The external guide sequence transcripts bind to the targetedmRNA species by the formation of base pairs between the mRNA and thecomplementary external guide sequences, thus promoting cleavage of mRNAby RNase P at the nucleotide located at the 5′-side of the base-pairedregion.

In general, the dosage of a composition comprising a therapeutic vectorhaving a Zcytor14 nucleotide sequence, such as a recombinant virus, willvary depending upon such factors as the subject's age, weight, height,sex, general medical condition and previous medical history. Suitableroutes of administration of therapeutic vectors include intravenousinjection, intraarterial injection, intraperitoneal injection,intramuscular injection, intratumoral injection, and injection into acavity that contains a tumor. As an illustration, Horton et al., Proc.Nat'l Acad. Sci. USA 96:1553 (1999), demonstrated that intramuscularinjection of plasmid DNA encoding interferon-α produces potent antitumoreffects on primary and metastatic tumors in a murine model.

A composition comprising viral vectors, non-viral vectors, or acombination of viral and non-viral vectors of the present invention canbe formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby vectors or viruses are combined in amixture with a pharmaceutically acceptable carrier. As noted above, acomposition, such as phosphate-buffered saline is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient subject. Other suitable carriers are well-knownto those in the art (see, for example, Remington's PharmaceuticalSciences, 19th Ed. (Mack Publishing Co. 1995), and Gilman's thePharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co.1985)).

For purposes of therapy, a therapeutic gene expression vector, or arecombinant virus comprising such a vector, and a pharmaceuticallyacceptable carrier are administered to a subject in a therapeuticallyeffective amount. A combination of an expression vector (or virus) and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient subject. For example, an agent used to treat inflammation isphysiologically significant if its presence alleviates the inflammatoryresponse.

When the subject treated with a therapeutic gene expression vector or arecombinant virus is a human, then the therapy is preferably somaticcell gene therapy. That is, the preferred treatment of a human with atherapeutic gene expression vector or a recombinant virus does notentail introducing into cells a nucleic acid molecule that can form partof a human germ line and be passed onto successive generations (i.e.,human germ line gene therapy).

14. Production of Transgenic Mice

Transgenic mice can be engineered to over-express the Zcytor14 gene inall tissues or under the control of a tissue-specific ortissue-preferred regulatory element. These over-producers of Zcytor14can be used to characterize the phenotype that results fromover-expression, and the transgenic animals can serve as models forhuman disease caused by excess Zcytor14. Transgenic mice thatover-express Zcytor14 also provide model bioreactors for production ofZcytor14, such as soluble Zcytor14, in the milk or blood of largeranimals. Methods for producing transgenic mice are well-known to thoseof skill in the art (see, for example, Jacob, “Expression and Knockoutof Interferons in Transgenic Mice,” in Overexpression and Knockout ofCytokines in Transgenic Mice, Jacob (ed.), pages 111-124 (AcademicPress, Ltd. 1994), Monastersky and Robl (eds.), Strategies in TransgenicAnimal Science (ASM Press 1995), and Abbud and Nilson, “RecombinantProtein Expression in Transgenic Mice,” in Gene Expression Systems:Using Nature for the Art of Expression, Fernandez and Hoeffler (eds.),pages 367-397 (Academic Press, Inc. 1999)).

For example, a method for producing a transgenic mouse that expresses aZcytor14 gene can begin with adult, fertile males (studs) (B6C3fl, 2-8months of age (Taconic Farms, Germantown, N.Y.)), vasectomized males(duds) (B6D2fl, 2-8 months, (Taconic Farms)), prepubescent fertilefemales (donors) (B6C3fl, 4-5 weeks, (Taconic Farms)) and adult fertilefemales (recipients) (B6D2fl, 2-4 months, (Taconic Farms)). The donorsare acclimated for one week and then injected with approximately 8IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company;St. Louis, Mo.) I.P., and 46-47 hours later, 8 IU/mouse of humanChorionic Gonadotropin (hCG (Sigma)) I.P. to induce superovulation.Donors are mated with studs subsequent to hormone injections. Ovulationgenerally occurs within 13 hours of hCG injection. Copulation isconfirmed by the presence of a vaginal plug the morning followingmating.

Fertilized eggs are collected under a surgical scope. The oviducts arecollected and eggs are released into urinanalysis slides containinghyaluronidase (Sigma). Eggs are washed once in hyaluronidase, and twicein Whitten's W640 medium (described, for example, by Menino andO'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs, Zygote4:129 (1996)) that has been incubated with 5% CO₂, 5% O₂, and 90% N₂ at37° C. The eggs are then stored in a 37° C./5% CO₂ incubator untilmicroinjection.

Ten to twenty micrograms of plasmid DNA containing a Zcytor14 encodingsequence is linearized, gel-purified, and resuspended in 10 mM Tris-HCl(pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10nanograms per microliter for microinjection. For example, the Zcytor14encoding sequences can encode a polypeptide comprising amino acidresidues 21 to 452 of SEQ ID NO:2.

Plasmid DNA is microinjected into harvested eggs contained in a drop ofW640 medium overlaid by warm, CO₂-equilibrated mineral oil. The DNA isdrawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary), and injected into individual eggs. Eachegg is penetrated with the injection needle, into one or both of thehaploid pronuclei.

Picoliters of DNA are injected into the pronuclei, and the injectionneedle withdrawn without coming into contact with the nucleoli. Theprocedure is repeated until all the eggs are injected. Successfullymicroinjected eggs are transferred into an organ tissue-culture dishwith pre-gassed W640 medium for storage overnight in a 37° C./5% CO₂incubator.

The following day, two-cell embryos are transferred into pseudopregnantrecipients. The recipients are identified by the presence of copulationplugs, after copulating with vasectomized duds. Recipients areanesthetized and shaved on the dorsal left side and transferred to asurgical microscope. A small incision is made in the skin and throughthe muscle wall in the middle of the abdominal area outlined by theribcage, the saddle, and the hind leg, midway between knee and spleen.The reproductive organs are exteriorized onto a small surgical drape.The fat pad is stretched out over the surgical drape, and a babyserrefine (Roboz, Rockville, Md.) is attached to the fat pad and lefthanging over the back of the mouse, preventing the organs from slidingback in.

With a fine transfer pipette containing mineral oil followed byalternating W640 and air bubbles, 12-17 healthy two-cell embryos fromthe previous day's injection are transferred into the recipient. Theswollen ampulla is located and holding the oviduct between the ampullaand the bursa, a nick in the oviduct is made with a 28 g needle close tothe bursa, making sure not to tear the ampulla or the bursa.

The pipette is transferred into the nick in the oviduct, and the embryosare blown in, allowing the first air bubble to escape the pipette. Thefat pad is gently pushed into the peritoneum, and the reproductiveorgans allowed to slide in. The peritoneal wall is closed with onesuture and the skin closed with a wound clip. The mice recuperate on a37° C. slide warmer for a minimum of four hours.

The recipients are returned to cages in pairs, and allowed 19-21 daysgestation. After birth, 19-21 days postpartum is allowed before weaning.The weanlings are sexed and placed into separate sex cages, and a 0.5 cmbiopsy (used for genotyping) is snipped off the tail with cleanscissors.

Genomic DNA is prepared from the tail snips using, for example, a QIAGENDNEASY kit following the manufacturer's instructions. Genomic DNA isanalyzed by PCR using primers designed to amplify a Zcytor14 gene or aselectable marker gene that was introduced in the same plasmid. Afteranimals are confirmed to be transgenic, they are back-crossed into aninbred strain by placing a transgenic female with a wild-type male, or atransgenic male with one or two wild-type female(s). As pups are bornand weaned, the sexes are separated, and their tails snipped forgenotyping.

To check for expression of a transgene in a live animal, a partialhepatectomy is performed. A surgical prep is made of the upper abdomendirectly below the zyphoid process. Using sterile technique, a small1.5-2 cm incision is made below the sternum and the left lateral lobe ofthe liver exteriorized. Using 4-0 silk, a tie is made around the lowerlobe securing it outside the body cavity. An atraumatic clamp is used tohold the tie while a second loop of absorbable Dexon (American Cyanamid;Wayne, N.J.) is placed proximal to the first tie. A distal cut is madefrom the Dexon tie and approximately 100 mg of the excised liver tissueis placed in a sterile petri dish. The excised liver section istransferred to a 14 ml polypropylene round bottom tube and snap frozenin liquid nitrogen and then stored on dry ice. The surgical site isclosed with suture and wound clips, and the animal's cage placed on a37° C. heating pad for 24 hours post operatively. The animal is checkeddaily post operatively and the wound clips removed 7-10 days aftersurgery. The expression level of Zcytor14 mRNA is examined for eachtransgenic mouse using an RNA solution hybridization assay or polymerasechain reaction.

In addition to producing transgenic mice that over-express Zcytor14, itis useful to engineer transgenic mice with either abnormally low or noexpression of the gene. Such transgenic mice provide useful models fordiseases associated with a lack of Zcytor14. As discussed above,Zcytor14 gene expression can be inhibited using anti-sense genes,ribozyme genes, or external guide sequence genes. To produce transgenicmice that under-express the Zcytor14 gene, such inhibitory sequences aretargeted to Zcytor14 mRNA. Methods for producing transgenic mice thathave abnormally low expression of a particular gene are known to thosein the art (see, for example, Wu et al., “Gene Underexpression inCultured Cells and Animals by Antisense DNA and RNA Strategies,” inMethods in Gene Biotechnology, pages 205-224 (CRC Press 1997)).

An alternative approach to producing transgenic mice that have little orno Zcytor14 gene expression is to generate mice having at least onenormal Zcytor14 allele replaced by a nonfunctional Zcytor14 gene. Onemethod of designing a nonfunctional Zcytor14 gene is to insert anothergene, such as a selectable marker gene, within a nucleic acid moleculethat encodes Zcytor14. Standard methods for producing these so-called“knockout mice” are known to those skilled in the art (see, for example,Jacob, “Expression and Knockout of Interferons in Transgenic Mice,” inOverexpression and Knockout of Cytokines in Transgenic Mice, Jacob(ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu et al., “NewStrategies for Gene Knockout,” in Methods in Gene Biotechnology, pages339-365 (CRC Press 1997)).

The present invention, thus generally described, will be understood morereadily by reference to the following example, which is provided by wayof illustration and is not intended to be limiting of the presentinvention.

EXAMPLE 1 Expression of the Zcytor14 Gene

Northern analyses were performed using Human Multiple Tissue Blots(CLONTECH Laboratories, Inc., Palo Alto, Calif.). Two probes weregenerated from gel purified PCR products. The first probe was made usingZC21798 (5′ CGG CGT GGT GGT CTT GCT CTT 3′; SEQ ID NO:8) and ZC21808 (5′TCC CGT CCC CCG CCC CAG GTC 3′; SEQ ID NO:9) as primers. The probe was aradioactively labeled using the Multiprime labeling kit from Amersham(Arlington Heights, Ill.) according to the manufacturer's protocol. Theprobe was purified using a NUCTRAP push column (STRATAGENE, La Jolla,Calif.). EXPRESSHYB (CLONTECH) solution was used for theprehybridization and hybridization solutions for the northern blots.Hybridization took place overnight at 65□C. Following hybridization, theblots were washed for 30 minutes each in solutions that contained 0.1%SDS and SSC as follows: twice in 2×SSC at room temperature, three timesin 0.1×SSC at 50□C., once in 0.1×SSC at 55□C., and once in 0.1×SSC at65□C. The results demonstrated the Zcytor14 gene is strongly expressedin thyroid, adrenal gland, prostate, and liver tissues, and expressed toa lesser extent in heart, small intestine, stomach, and trachea tissues.In contrast, there is little or no expression in brain, placenta, lung,skeletal muscle, kidney, pancreas, spleen, thymus, testis, ovary, colon,peripheral blood leukocytes, spinal cord, lymph node, and bone marrow.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A vector, comprising an isolated polynucleotide that encodes aZcytor14 polypeptide, wherein the nucleic acid molecule is selected fromthe group consisting of: (a) a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:3, (b) a nucleic acid molecule encodingan amino acid sequence that comprises either amino acid residues 21 to677 of SEQ ID NO:2 or amino acid residues 21 to 673 of SEQ ID NO:10, and(c) a nucleic acid molecule that remains hybridized following stringentwash conditions to a nucleic acid molecule comprising the nucleotidesequence of nucleotides 214 to 2184 of SEQ ID NO:1, or the complement ofnucleotides 214 to 2184 of SEQ ID NO:1.
 2. The vector of claim 1,wherein the polynucleotide is selected form the group consisting of: SEQID NO:1 and SEQ ID NO:3.
 3. The vector of claim 1, wherein thepolynucleotide encodes a polypeptide comprising an amino acid sequenceselected from the group consisting of: (a) amino acid residues 21 to 452of SEQ ID NO:2, (b) amino acid residues 21 to 435 of SEQ ID NO:10, (c)amino acid residues 21 to 677 of SEQ ID NO:2, and (d) amino acidresidues 1 to 692 of SEQ ID NO:2.
 4. The vector of claim 1, wherein theencoded polypeptide consists of an amino acid sequence selected from thegroup consisting of: (a) amino acid residues 21 to 452 of SEQ ID NO:2,(b) amino acid residues 21 to 435 of SEQ ID NO:10, (c) amino acidresidues 21 to 677 of SEQ ID NO:2, and (d) amino acid residues 1 to 692of SEQ ID NO:2.
 5. The vector of claim 1, wherein the vector furthercomprises a transcription promoter, and a transcription terminator,wherein the promoter is operably linked with the nucleic acid molecule,and wherein the nucleic acid molecule is operably linked with thetranscription terminator.
 6. The vector of claim 4, wherein the vectorfurther comprises a transcription promoter, and a transcriptionterminator, wherein the promoter is operably linked with the nucleicacid molecule, and wherein the nucleic acid molecule is operably linkedwith the transcription terminator.
 7. A recombinant virus, comprisingthe expression vector of claim
 5. 8. A recombinant virus, comprising theexpression vector of claim
 6. 9. A recombinant host cell comprising thevector of claim 5, wherein the host cell is selected from the groupconsisting of bacterium, yeast cell, fungal cell, insect cell, mammaliancell, and plant cell.
 10. A recombinant host cell comprising the vectorof claim 6, wherein the host cell is selected from the group consistingof bacterium, yeast cell, fungal cell, insect cell, mammalian cell, andplant cell.
 11. A method of using the expression vector of claim 5 toproduce Zcytor14 protein, the method comprising the step of culturingrecombinant host cells that comprise the expression vector and thatproduce the Zcytor14 protein.
 12. A method of using the expressionvector of claim 6 to produce Zcytor14 protein, the method comprising thestep of culturing recombinant host cells that comprise the expressionvector and that produce the Zcytor14 protein.